Isolated human drug-metabolizing proteins, nucleic acid molecules encoding human drug-metabolizing proteins, and uses thereof

ABSTRACT

The present invention provides amino acid sequences of peptides that are encoded by genes within the human genome, the drug-metabolizing enzyme peptides of the present invention. The present invention specifically provides isolated peptide and nucleic acid molecules, methods of identifying orthologs and paralogs of the drug-metabolizing enzyme peptides, and methods of identifying modulators of the drug-metabolizing enzyme peptides.

FIELD OF THE INVENTION

[0001] The present invention is in the field of drug-metabolizingproteins that are related to the UDP-glycosyltransferasedrug-metabolizing enzyme subfamily, recombinant DNA molecules andprotein production. The present invention specifically provides noveldrug-metabolizing peptides and proteins and nucleic acid moleculesencoding such protein molecules, for use in the development of humantherapeutics and human therapeutic development.

BACKGROUND OF THE INVENTION

[0002] Drug-Metabolizing Proteins

[0003] Induction of drug-metabolizing enzymes (“DMEs”) is a commonbiological response to xenobiotics, the mechanisms and consequences ofwhich are important in academic, industrial, and regulatory areas ofpharmacology and toxicology.

[0004] For most drugs, drug-metabolizing enzymes determine how long andhow much of a drug remains in the body. Thus, developers of drugsrecognize the importance of characterizing a drug candidate'sinteraction with these enzymes. For example, polymorphisms of thedrug-metabolizing enzyme CYP2D6, a member of the cytochrome p450 (“CYP”)superfamily, yield phenotypes of slow or ultra-rapid metabolizers of awide spectrum of drugs including antidepressants, antipsychotics,beta-blockers, and antiarrhythmics. Such abnormal rates of drugmetabolism can lead to drug ineffectiveness or to systemic accumulationand toxicity.

[0005] For pharmaceutical scientists developing a candidate drug, it isimportant know as early as possible in the design phase which enzymesmetabolize the drug candidate and the speed with which they do it.Historically, the enzymes on a drug's metabolic pathway were determinedthrough metabolism studies in animals, but this approach has now beenlargely supplanted by the use of human tissues or cloneddrug-metabolizing enzymes to provide insights into the specific role ofindividual forms of these enzymes. Using these tools, the qualitativeand quantitative fate of a drug candidate can be predicted prior to itsfirst administration to humans. As a consequence, the selection andoptimization of desirable characteristics of metabolism are possibleearly in the development process, thus avoiding unanticipated toxicityproblems and associated costs subsequent to the drug's clinicalinvestigation. Moreover, the effect of one drug on another's dispositioncan be inferred.

[0006] Known drug-metabolizing enzymes include the cytochrome p450(“CYP”) superfamily, N-acetyl transferases (“NAT”), UDP-glucuronosyltransferases (“UGT”), methyl transferases, alcohol dehydrogenase(“ADH”), aldehyde dehydrogenase (“ALDH”), dihydropyrimidinedehydrogenase (“DPD”), NADPH:quinone oxidoreductase (“NQO” or “DTdiaphorase”), catecho O-methyltransferase (“COMT”), glutathioneS-transferase (“GST”), histamine methyltransferase (“HMT”),sulfotransferases (“ST”), thiopurine methyltransferase (“TPMT”), andepoxide hydroxylase. Drug-metabolizing enzymes are generally classifiedinto two phases according to their metabolic function. Phase I enzymescatalyze modification of functional groups, and phase II enzymescatalyze conjugation with endogenous substituents. These classificationsshould not be construed as exclusive nor exhaustive, as other mechanismsof drug metabolism have been discovered. For example, the use of activetransport mechanisms been characterized as part of the process ofdetoxification.

[0007] Phase I reactions include catabolic processes such as deaminationof aminases, hydrolysis of esters and amides, conjugation reactionswith, for example, glycine or sulfate, oxidation by the cytochrome p450oxidation/reduction enzyme system and degradation in the fatty acidpathway. Hydrolysis reactions occur mainly in the liver and plasma by avariety of non-specific hydrolases and esterases. Both deaminases andamidases, also localized in the liver and serum, carry out a large partof the catabolic process. Reduction reactions occur mainlyintracellularly in the endoplasmic reticulum.

[0008] Phase II enzymes detoxify toxic substances by catalyzing theirconjugation with water-soluble substances, thus increasing toxins'solubility in water and increasing their rate of excretion.Additionally, conjugation reduces the toxins' biological reactivity.Examples of phase II enzymes include glutathione S-transferases andUDP-glucuronosyl transferases, which catalyze conjugation to glutathioneand glucuronic acid, respectively. Transferases perform conjugationreactions mainly in the kidneys and liver.

[0009] The liver is the primary site of elimination of most drugs,including psychoactive drugs, and contains a plurality of both phase Iand phase II enzymes that oxidize or conjugate drugs, respectively.

[0010] Physicians currently prescribe drugs and their dosages based on apopulation average and fail to take genetic variability into account.The variability between individuals in drug metabolism is usually due toboth genetic and environmental factors, in particular, how thedrug-metabolizing enzymes are controlled. With certain enzymes, thegenetic component predominates and variability is associated withvariants of the normal, wild-type enzyme.

[0011] Most drug-metabolizing enzymes exhibit clinically relevantgenetic polymorphisms. Essentially all of the major human enzymesresponsible for modification of functional groups or conjugation withendogenous subsituents exhibit common polymorphisms at the genomiclevel. For example, polymorphisms expressing a non-functioning variantenzyme results in a sub-group of patients in the population who are moreprone to the concentration-dependent effects of a drug. This sub-groupof patients may show toxic side effects to a dose of drug that isotherwise without side effects in the general population. Recentdevelopment in genotyping allows identification of affected individuals.As a result, their atypical metabolism and likely response to a drugmetabolized by the affected enzyme can be understood and predicted, thuspermitting the physician to adjust the dose of drug they receive toachieve improved therapy.

[0012] A similar approach is also becoming important in identifying riskfactors associated with the development of various cancers. This isbecause the enzymes involved in drug metabolism are also responsible forthe activation and detoxification of chemical carcinogens. Specifically,the development of neoplasia is regulated by a balance between phase Ienzymes, which activate carcinogens, and phase II enzymes, whichdetoxify them. Accordingly, an individual's susceptibility to canceroften involves the balance between these two processes, which is, inpart, genetically determined and can be screened by suitable genotypingtests. Higher induction of phase I enzymes compared to phase II enzymesresults in the generation of large amounts of electrophiles and reactiveoxygen species and may cause DNA and membrane damage and other adverseeffects leading to neoplasia. Conversely, higher levels of phase IIenzyme expression can protect cells from various chemical compounds.

[0013] Abnormal activity of drug-metabolizing enzymes has beenimplicated in a range of human diseases, including cancer, Parkinson'sdisease, myetonic dystrophy, and developmental defects.

[0014] Cytochrome p450

[0015] An example of a phase I drug-metabolizing enzyme is thecytochrome p450 (“CYP”) superfamily, the members of which comprise themajor drug-metabolizing enzymes expressed in the liver. The CYPsuperfamily comprises heme proteins which catalyze the oxidation anddehydrogenation of a number of endogenous and exogenous lipophiliccompounds. The CYP superfamily has immense diversity in its functions,with hundreds of isoforms in many species catalyzing many types ofchemical reactions. The CYP superfamily comprises at least 30 relatedenzymes, which are divided into different families according to theiramino acid homology. Examples of CYP families include CYP families 1, 2,3 and 4, which comprise endoplasmic reticulum proteins responsible forthe metabolism of drugs and other xenobiotics. Approximately 10-15individual gene products within these four families metabolize thousandsof structurally diverse compounds. It is estimated that collectively theenzymes in the CYP superfamily participate in the metabolism of greaterthan 80% of all available drugs used in humans. For example, the CYP 1Asubfamily comprises CYP 1A2, which metabolizes several widely useddrugs, including acetaminophen, amitriptyline, caffeine, clozapine,haloperidol, imipramine, olanzapine, ondansetron, phenacetin,propafenone, propranolol, tacrine, theophylline, verapamil. In addition,CYP enzymes play additional roles in the metabolism of some endogenoussubstrates including prostaglandins and steroids.

[0016] Some CYP enzymes exist in a polymorphic form, meaning that asmall percentage of the population possesses mutant genes that alter theactivity of the enzyme, usually by diminishing or abolishing activity.For example, a genetic polymorphism has been well characterized with theCYP 2C19 and CYP 2D6 genes. Substrates of CYP 2C19 include clomipramine,diazepam, imipramine, mephenytoin, moclobemide, omeprazole, phenytoin,propranolol, and tolbutamide. Substrates of CYP 2D6 include alprenolol,amitriptyline, chlorpheniramine, clomipramine, codeine, desipramine,dextromethorphan, encainide, fluoxetine, haloperidol, imipramine,indoramin, metoprolol, nortriptyline, ondansetron, oxycodone,paroxetine, propranolol, and propafenone. Polymorphic variants of thesegenes metabolize these substrates at different rates, which can effect apatient's effective therapeutic dosage.

[0017] While the substrate specificity of CYPs must be very broad toaccommodate the metabolism of all of these compounds, each individualCYP gene product has a narrower substrate specificity defined by itsbinding and catalytic sites. Drug metabolism can thereby be regulated bychanges in the amount or activity of specific CYP gene products. Methodsof CYP regulation include genetic differences in the expression of CYPgene products (i.e., genetic polymorphisms), inhibition of CYPmetabolism by other xenobiotics that also bind to the CYP, and inductionof certain CYPs by the drug itself or other xenobiotics. Inhibition andinduction of CYPs is one of the most common mechanisms of adverse druginteractions. For example, the CYP3A subfamily is involved in clinicallysignificant drug interactions involving nonsedating antihistamines andcisapride that may result in cardiac dysrhythmias. In another example,CYP3A4 and CYP1A2 enzymes are involved in drug interactions involvingtheophylline. In yet another example, CYP2D6 is responsible for themetabolism of many psychotherapeutic agents. Additionallly, CYP enzymesmetabolize the protease inhibitors used to treat patients infected withthe human immunodeficiency virus. By understanding the unique functionsand characteristics of these enzymes, physicians may better anticipateand manage drug interactions and may predict or explain an individual'sresponse to a particular therapeutic regimen.

[0018] Examples of reactions catalyzed by the CYP superfamily includeperoxidative reactions utilizing peroxides as oxygen donors inhydroxylation reactions, as substrates for reductive beta-scission, andas peroxyhemiacetal intermediates in the cleavage of aldehydes toformate and alkenes. Lipid hydroperoxides undergo reductivebeta-cleavage to give hydrocarbons and aldehydic acids. One of theseproducts, trans-4-hydroxynonenal, inactivates CYP, particularlyalcohol-inducible 2E1, in what may be a negative regulatory process.Although a CYP iron-oxene species is believed to be the oxygen donor inmost hydroxylation reactions, an iron-peroxy species is apparentlyinvolved in the deformylation of many aldehydes with desaturation of theremaining structure, as in aromatization reactions.

[0019] Examples of drugs with oxidative metabolism associated with CYPenzymes include acetaminophen, alfentanil, alprazolam, alprenolol,amiodarone, amitriptyline, astemizole, buspirone caffeine,carbamazepine, chlorpheniramine, cisapride, clomipramine, clomipramine,clozapine, codeine, colchicine, cortisol, cyclophosphamide,cyclosporine, dapsone, desipramine, dextromethorphan, diazepam,diclofenac, diltiazem, encainide, erythromycin, estradiol, felodipine,fluoxetine, fluvastatin, haloperidol, ibuprofen, imipramine, indinavir,indomethacin, indoramin, irbesartan, lidocaine, losartan, macrolideantibiotics, mephenytoin, methadone, metoprolol, mexilitene, midazolam,moclobemide, naproxen, nefazodone, nicardipine, nifedipine,nitrendipine, nortriptyline, olanzapine, omeprazole, ondansetron,oxycodone, paclitaxel, paroxetine, phenacetin, phenytoin, piroxicam,progesterone, propafenone, propranolol, quinidine, ritonavir,saquinavir, sertraline, sildenafil, S-warfarin, tacrine, tamoxifen,tenoxicam, terfenadine, testosterone, theophylline, timolol,tolbutamide, triazolam, verapamil, and vinblastine.

[0020] Abnormal activity of phase I enzymes has been implicated in arange of human diseases. For example, enhanced CYP2D6 activity has beenrelated to malignancies of the bladder, liver, pharynx, stomach andlungs, whereas decreased CYP2D activity has been linked to an increasedrisk of Parkinson's disease. Other syndromes and developmental defectsassociated with deficiencies in the CYP superfamily includecerebrotendinous xanthomatosis, adrenal hyperplasia, gynecomastia, andmyetonic dystrophy.

[0021] The CYP superfamily a major target for drug action anddevelopment. Accordingly, it is valuable to the field of pharmaceuticaldevelopment to identify and characterize previously unknown members ofthe CYP superfamily.

[0022] UDP-Glycosyltransferases

[0023] Potential drug interactions involving phase II metabolism areincreasingly being recognized. An important group of phase II enzymesinvolved in drug metabolism are the glucuronosyltransferases, especiallythe UDP-glycosyltransferase (alternatively referred to asUDP-glucuronosyltransferase) (“UGT”) superfamily. The novel humanprotein, and encoding gene, provided by the present invention shows thehighest degree of similarity to UGT2B11. Members of the UGT2B family areimportant for conjugating glucuronic acid to bile salts and steroidhormones, as well as to phenol derivatives, bilirubin, and fatty acids.For a further review of UGT2B11, see Beaulieu et al., Biochem BiophysRes Commun 1998 July 9;248(1):44-50 and Jin et al., Biochem. Biophys.Res. Commun. 194: 496-503, 1993.

[0024] Members of the UGT superfamily catalyze the enzymatic addition ofUDP glucuronic acid as a sugar donor to fat-soluble chemicals, a processthat increases their solubility in water and increases their rate ofexcretion. In mammals, glucuronic acid is the main sugar that is used toprevent the accumulation of waste products of metabolism and fat-solublechemicals from the environment to toxic levels in the body. Bothinducers and inhibitors of glucuronosyltransferases are known and havethe potential to affect the plasma concentration and actions ofimportant drugs, including psychotropic drugs.

[0025] The UGT superfamily comprises several families of enzymes inseveral species defined with a nomenclature similar to that used todefine members of the CYP superfamily. In animals, yeast, plants andbacteria there are at least 110 distinct known members of the UGTsuperfamily. As many as 33 families have been defined, with threefamilies identified in humans. Different UGT families are defined ashaving <45% amino acid sequence homology; within subfamilies there isapproximately 60% homology. The members of the UGT superfamily are partof a further superfamily of UDP glycosyltransferases found in animals,plants and bacteria.

[0026] The role of phase II enzymes, and of UGT enzymes in particular,is being increasingly recognized as important in psychopharmacology. UGTenzymes conjugate many important psychotropic drugs and are an importantsource of variability in drug response and drug interactions. Forexample, the benzodiazepines lorazepam, oxazepam, and temazepam undergophase II reactions exclusively before being excreted into the urine.

[0027] Phase II enzymes metabolize and detoxify hazardous substances,such as carcinogens. The expression of genes encoding phase II enzymesis known to be up-regulated by hundreds of agents. For example, oltiprazis known to up-regulate phase II enzyme expression. Studies havedemonstrated protection from the cancer-causing effects of carcinogenswhen selected phase II enzyme inducers are administered prior to thecarcinogens. The potential use of phase II enzyme inducers in humans forprevention of cancers related to exposure to carcinogens has promptedstudies aimed at understanding their molecular effects. Currentbiochemical and molecular biological research methodologies can be usedto identify and characterize selective phase II enzyme inducers andtheir targets. Identification of genes responding to cancerchemopreventive agents will facilitate studies of their basic mechanismand provide insights about the relationship between gene regulation,enzyme polymorphism, and carcinogen detoxification.

[0028] Examples of drugs with conjugative metabolism associated with UGTenzymes include amitriptyline, buprenorphine, chlorpromazine, clozapine,codeine, cyproheptadine, dihydrocodeine, doxepin, imipramine,lamotrigine, lorazepam, morphine, nalorphine, naltrexone, temazepam, andvalproate.

[0029] Abnormal activity of phase II enzymes has been implicated in arange of human diseases. For example, Gilbert syndrome is an autosomaldominant disorder caused by mutation in the UGT1 gene, and mutations inthe UGT1A1 enzyme have been demonstrated to be responsible forCrigler-Najjar syndrome.

[0030] Drug-metabolizing enzymes, particularly members of theUDP-glycosyltransferase drug-metabolizing enzyme subfamily, are a majortarget for drug action and development. Accordingly, it is valuable tothe field of pharmaceutical development to identify and characterizepreviously unknown members of this subfamily of drug-metabolizingproteins. The present invention advances the state of the art byproviding previously unidentified human drug-metabolizing proteins thathave homology to members of the UDP-glycosyltransferasedrug-metabolizing enzyme subfamily.

SUMMARY OF THE INVENTION

[0031] The present invention is based in part on the identification ofamino acid sequences of human drug-metabolizing enzyme peptides andproteins that are related to the UDP-glycosyltransferasedrug-metabolizing enzyme subfamily, as well as allelic variants andother mammalian orthologs thereof. These unique peptide sequences, andnucleic acid sequences that encode these peptides, can be used as modelsfor the development of human therapeutic targets, aid in theidentification of therapeutic proteins, and serve as targets for thedevelopment of human therapeutic agents that modulate drug-metabolizingenzyme activity in cells and tissues that express the drug-metabolizingenzyme. Experimental data as provided in FIG. 1 indicates expression inhumans in the eye, kidney, hepatocellular carcinoma, and a mixedbrain/heart/kidney/lung/spleen/testis/leukocyte sample.

DESCRIPTION OF THE FIGURE SHEETS

[0032]FIG. 1 provides the nucleotide sequence of a cDNA molecule thatencodes the drug-metabolizing enzyme protein of the present invention.(SEQ ID NO:1) In addition, structure and functional information isprovided, such as ATG start, stop and tissue distribution, whereavailable, that allows one to readily determine specific uses ofinventions based on this molecular sequence. Experimental data asprovided in FIG. 1 indicates expression in humans in the eye, kidney,hepatocellular carcinoma, and a mixedbrain/heart/kidney/lung/spleen/testis/leukocyte sample.

[0033]FIG. 2 provides the predicted amino acid sequence of thedrug-metabolizing enzyme of the present invention. (SEQ ID NO:2) Inaddition structure and functional information such as protein family,function, and modification sites is provided where available, allowingone to readily determine specific uses of inventions based on thismolecular sequence.

DETAILED DESCRIPTION OF THE INVENTION

[0034] General Description

[0035] The present invention is based on the sequencing of the humangenome. During the sequencing and assembly of the human genome, analysisof the sequence information revealed previously unidentified fragmentsof the human genome that encode peptides that share structural and/orsequence homology to protein/peptide/domains identified andcharacterized within the art as being a drug-metabolizing enzyme proteinor part of a drug-metabolizing enzyme protein and are related to theUDP-glycosyltransferase drug-metabolizing enzyme subfamily. Utilizingthese sequences, additional genomic sequences were assembled andtranscript and/or cDNA sequences were isolated and characterized. Basedon this analysis, the present invention provides amino acid sequences ofhuman drug-metabolizing enzyme peptides and proteins that are related tothe UDP-glycosyltransferase drug-metabolizing enzyme subfamily, andnucleic acid sequences in the form of cDNA/transcript sequences thatencode these drug-metabolizing enzyme peptides and proteins, tissuedistribution of expression, and information about the closest art knownprotein/peptide/domain that has structural or sequence homology to thedrug-metabolizing enzyme of the present invention.

[0036] In addition to being previously unknown, the peptides that areprovided in the present invention are selected based on their ability tobe used for the development of commercially important products andservices. Specifically, the present peptides are selected based onhomology and/or structural relatedness to known drug-metabolizing enzymeproteins of the UDP-glycosyltransferase drug-metabolizing enzymesubfamily and the expression pattern observed. Experimental data asprovided in FIG. 1 indicates expression in humans in the eye, kidney,hepatocellular carcinoma, and a mixedbrain/heart/kidney/lung/spleen/testis/leukocyte sample. The art hasclearly established the commercial importance of members of this familyof proteins and proteins that have expression patterns similar to thatof the present gene. Some of the more specific features of the peptidesof the present invention, and the uses thereof, are described herein,particularly in the Background of the Invention and in the annotationprovided in the Figures, and/or are known within the art for each of theknown UDP-glycosyltransferase family or subfamily of drug-metabolizingenzyme proteins.

[0037] Specific Embodiments

[0038] Peptide Molecules

[0039] The present invention provides nucleic acid sequences that encodeprotein molecules that have been identified as being members of thedrug-metabolizing enzyme family of proteins and are related to theUDP-glycosyltransferase drug-metabolizing enzyme subfamily (proteinsequences are provided in FIG. 2, transcript/cDNA sequences are providedin FIG. 1). The peptide sequences provided in FIG. 2, as well as theobvious variants described herein, will be referred herein as thedrug-metabolizing enzyme peptides of the present invention,drug-metabolizing enzyme peptides, or peptides/proteins of the presentinvention.

[0040] The present invention provides isolated peptide and proteinmolecules that consist of, consist essentially of, or comprise the aminoacid sequences of the drug-metabolizing enzyme peptides disclosed inFIG. 2, (encoded by the nucleic acid molecule shown in FIG. 1,transcript/cDNA), as well as all obvious variants of these peptides thatare within the art to make and use. Some of these variants are describedin detail below.

[0041] As used herein, a peptide is said to be “isolated” or “purified”when it is substantially free of cellular material or free of chemicalprecursors or other chemicals. The peptides of the present invention canbe purified to homogeneity or other degrees of purity. The level ofpurification will be based on the intended use. The critical feature isthat the preparation allows for the desired function of the peptide,even if in the presence of considerable amounts of other components (thefeatures of an isolated nucleic acid molecule is discussed below).

[0042] In some uses, “substantially free of cellular material” includespreparations of the peptide having less than about 30% (by dry weight)other proteins (i.e., contaminating protein), less than about 20% otherproteins, less than about 10% other proteins, or less than about 5%other proteins. When the peptide is recombinantly produced, it can alsobe substantially free of culture medium, i.e., culture medium representsless than about 20% of the volume of the protein preparation.

[0043] The language “substantially free of chemical precursors or otherchemicals” includes preparations of the peptide in which it is separatedfrom chemical precursors or other chemicals that are involved in itssynthesis. In one embodiment, the language “substantially free ofchemical precursors or other chemicals” includes preparations of thedrug-metabolizing enzyme peptide having less than about 30% (by dryweight) chemical precursors or other chemicals, less than about. 20%chemical precursors or other chemicals, less than about 10% chemicalprecursors or other chemicals, or less than about 5% chemical precursorsor other chemicals.

[0044] The isolated drug-metabolizing enzyme peptide can be purifiedfrom cells that naturally express it, purified from cells that have beenaltered to express it (recombinant), or synthesized using known proteinsynthesis methods. Experimental data as provided in FIG. 1 indicatesexpression in humans in the eye, kidney, hepatocellular carcinoma, and amixed brain/heart/kidney/lung/spleen/testis/leukocyte sample. Forexample, a nucleic acid molecule encoding the drug-metabolizing enzymepeptide is cloned into an expression vector, the expression vectorintroduced into a host cell and the protein expressed in the host cell.The protein can then be isolated from the cells by an appropriatepurification scheme using standard protein purification techniques. Manyof these techniques are described in detail below.

[0045] Accordingly, the present invention provides proteins that consistof the amino acid sequences provided in FIG. 2 (SEQ ID NO:2), forexample, proteins encoded by the transcript/cDNA nucleic acid sequencesshown in FIG. 1 (SEQ ID NO:1). The amino acid sequence of such a proteinis provided in FIG. 2. A protein consists of an amino acid sequence whenthe amino acid sequence is the final amino acid sequence of the protein.

[0046] The present invention further provides proteins that consistessentially of the amino acid sequences provided in FIG. 2 (SEQ IDNO:2), for example, proteins encoded by the transcript/cDNA nucleic acidsequences shown in FIG. 1 (SEQ ID NO:1). A protein consists essentiallyof an amino acid sequence when such an amino acid sequence is presentwith only a few additional amino acid residues, for example from about 1to about 100 or so additional residues, typically from 1 to about 20additional residues in the final protein.

[0047] The present invention further provides proteins that comprise theamino acid sequences provided in FIG. 2 (SEQ ID NO:2), for example,proteins encoded by the transcript/cDNA nucleic acid sequences shown inFIG. 1 (SEQ ID NO:1). A protein comprises an amino acid sequence whenthe amino acid sequence is at least part of the final amino acidsequence of the protein. In such a fashion, the protein can be only thepeptide or have additional amino acid molecules, such as amino acidresidues (contiguous encoded sequence) that are naturally associatedwith it or heterologous amino acid residues/peptide sequences. Such aprotein can have a few additional amino acid residues or can compriseseveral hundred or more additional amino acids. The preferred classes ofproteins that are comprised of the drug-metabolizing enzyme peptides ofthe present invention are the naturally occurring mature proteins. Abrief description of how various types of these proteins can bemade/isolated is provided below.

[0048] The drug-metabolizing enzyme peptides of the present inventioncan be attached to heterologous sequences to form chimeric or fusionproteins. Such chimeric and fusion proteins comprise a drug-metabolizingenzyme peptide operatively linked to a heterologous protein having anamino acid sequence not substantially homologous to thedrug-metabolizing enzyme peptide. “Operatively linked” indicates thatthe drug-metabolizing enzyme peptide and the heterologous protein arefused in-frame. The heterologous protein can be fused to the N-terminusor C-terminus of the drug-metabolizing enzyme peptide.

[0049] In some uses, the fusion protein does not affect the activity ofthe drug-metabolizing enzyme peptide per se. For example, the fusionprotein can include, but is not limited to, enzymatic fusion proteins,for example beta-galactosidase fusions, yeast two-hybrid GAL fusions,poly-His fusions, MYC-tagged, HI-tagged and Ig fusions. Such fusionproteins, particularly poly-His fusions, can facilitate the purificationof recombinant drug-metabolizing enzyme peptide. In certain host cells(e.g., mammalian host cells), expression and/or secretion of a proteincan be increased by using a heterologous signal sequence.

[0050] A chimeric or fusion protein can be produced by standardrecombinant DNA techniques. For example, DNA fragments coding for thedifferent protein sequences are ligated together in-frame in accordancewith conventional techniques. In another embodiment, the fusion gene canbe synthesized by conventional techniques including automated DNAsynthesizers. Alternatively, PCR amplification of gene fragments can becarried out using anchor primers which give rise to complementaryoverhangs between two consecutive gene fragments which can subsequentlybe annealed and re-amplified to generate a chimeric gene sequence (seeAusubel et al., Current Protocols in Molecular Biology, 1992). Moreover,many expression vectors are commercially available that already encode afusion moiety (e.g., a GST protein). A drug-metabolizing enzymepeptide-encoding nucleic acid can be cloned into such an expressionvector such that the fusion moiety is linked in-frame to thedrug-metabolizing enzyme peptide.

[0051] As mentioned above, the present invention also provides andenables obvious variants of the amino acid sequence of the proteins ofthe present invention, such as naturally occurring mature forms of thepeptide, allelic/sequence variants of the peptides, non-naturallyoccurring recombinantly derived variants of the peptides, and orthologsand paralogs of the peptides. Such variants can readily be generatedusing art-known techniques in the fields of recombinant nucleic acidtechnology and protein biochemistry. It is understood, however, thatvariants exclude any amino acid sequences disclosed prior to theinvention.

[0052] Such variants can readily be identified/made using moleculartechniques and the sequence information disclosed herein. Further, suchvariants can readily be distinguished from other peptides based onsequence and/or structural homology to the drug-metabolizing enzymepeptides of the present invention. The degree of homology/identitypresent will be based primarily on whether the peptide is a functionalvariant or non-functional variant, the amount of divergence present inthe paralog family and the evolutionary distance between the orthologs.

[0053] To determine the percent identity of two amino acid sequences ortwo nucleic acid sequences, the sequences are aligned for optimalcomparison purposes (e.g., gaps can be introduced in one or both of afirst and a second amino acid or nucleic acid sequence for optimalalignment and non-homologous sequences can be disregarded for comparisonpurposes). In a preferred embodiment, at least 30%, 40%, 50%, 60%, 70%,80%, or 90% or more of the length of a reference sequence is aligned forcomparison purposes. The amino acid residues or nucleotides atcorresponding amino acid positions or nucleotide positions are thencompared. When a position in the first sequence is occupied by the sameamino acid residue or nucleotide as the corresponding position in thesecond sequence, then the molecules are identical at that position (asused herein amino acid or nucleic acid “identity” is equivalent to aminoacid or nucleic acid “homology”). The percent identity between the twosequences is a function of the number of identical positions shared bythe sequences, taking into account the number of gaps, and the length ofeach gap, which need to be introduced for optimal alignment of the twosequences.

[0054] The comparison of sequences and determination of percent identityand similarity between two sequences can be accomplished using amathematical algorithm. (Computational Molecular Biology, Lesk, A. M.,ed., Oxford University Press, New York, 1988; Biocomputing: Informaticsand Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993;Computer Analysis of Sequence Data, Part 1, Griffin, A. M., and Griffin,H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis inMolecular Biology, von Heinje, G., Academic Press, 1987; and SequenceAnalysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press,New York, 1991). In a preferred embodiment, the percent identity betweentwo amino acid sequences is determined using the Needleman and Wunsch(J. Mol. Biol. (48):444-453 (1970)) algorithm which has beenincorporated into the GAP program in the GCG software package (availableat http://www.gcg.com), using either a Blossom 62 matrix or a PAM250matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a lengthweight of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment, thepercent identity between two nucleotide sequences is determined usingthe GAP program in the GCG software package (Devereux, J., et al.,Nucleic Acids Res. 12(1):387 (1984)) (available at http://www.gcg.com),using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80and a length weight of 1, 2, 3, 4, 5, or 6. In another embodiment, thepercent identity between two amino acid or nucleotide sequences isdetermined using the algorithm of E. Myers and W. Miller (CABIOS,4:11-17 (1989)) which has been incorporated into the ALIGN program(version 2.0), using a PAM120 weight residue table, a gap length penaltyof 12 and a gap penalty of 4.

[0055] The nucleic acid and protein sequences of the present inventioncan further be used as a “query sequence” to perform a search againstsequence databases to, for example, identify other family members orrelated sequences. Such searches can be performed using the NBLAST andXBLAST programs (version 2.0) of Altschul, et al. (J. Mol. Biol.215:403-10 (1990)). BLAST nucleotide searches can be performed with theNBLAST program, score=100, wordlength=12 to obtain nucleotide sequenceshomologous to the nucleic acid molecules of the invention. BLAST proteinsearches can be performed with the XBLAST program, score=50,wordlength=3 to obtain amino acid sequences homologous to the proteinsof the invention. To obtain gapped alignments for comparison purposes,Gapped BLAST can be utilized as described in Altschul et al. (NucleicAcids Res. 25(17):3389-3402 (1997)). When utilizing BLAST and gappedBLAST programs, the default parameters of the respective programs (e.g.,XBLAST and NBLAST) can be used.

[0056] Full-length pre-processed forms, as well as mature processedforms, of proteins that comprise one of the peptides of the presentinvention can readily be identified as having complete sequence identityto one of the drug-metabolizing enzyme peptides of the present inventionas well as being encoded by the same genetic locus as thedrug-metabolizing enzyme peptide provided herein.

[0057] Allelic variants of a drug-metabolizing enzyme peptide canreadily be identified as being a human protein having a high degree(significant) of sequence homology/identity to at least a portion of thedrug-metabolizing enzyme peptide as well as being encoded by the samegenetic locus as the drug-metabolizing enzyme peptide provided herein.Genetic locus can readily be determined based on the sequenceinformation provided herein, and using sequence-based mapping methodswell known in the art. As used herein, two proteins (or a region of theproteins) have significant homology when the amino acid sequences aretypically at least about 70-80%, 80-90%, and more typically at leastabout 90-95% or more homologous. A significantly homologous amino acidsequence, according to the present invention, will be encoded by anucleic acid sequence that will hybridize to a drug-metabolizing enzymepeptide encoding nucleic acid molecule under stringent conditions asmore fully described below.

[0058] Paralogs of a drug-metabolizing enzyme peptide can readily beidentified as having some degree of significant sequencehomology/identity to at least a portion of the drug-metabolizing enzymepeptide, as being encoded by a gene from humans, and as having similaractivity or function. Two proteins will typically be considered paralogswhen the amino acid sequences are typically at least about 60% orgreater, and more typically at least about 70% or greater homologythrough a given region or domain. Such paralogs will be encoded by anucleic acid sequence that will hybridize to a drug-metabolizing enzymepeptide encoding nucleic acid molecule under moderate to stringentconditions as more fully described below.

[0059] Orthologs of a drug-metabolizing enzyme peptide can readily beidentified as having some degree of significant sequencehomology/identity to at least a portion of the drug-metabolizing enzymepeptide as well as being encoded by a gene from another organism.Preferred orthologs will be isolated from mammals, preferably primates,for the development of human therapeutic targets and agents. Suchorthologs will be encoded by a nucleic acid sequence that will hybridizeto a drug-metabolizing enzyme peptide encoding nucleic acid moleculeunder moderate to stringent conditions, as more fully described below,depending on the degree of relatedness of the two organisms yielding theproteins.

[0060] Non-naturally occurring variants of the drug-metabolizing enzymepeptides of the present invention can readily be generated usingrecombinant techniques. Such variants include, but are not limited todeletions, additions and substitutions in the amino acid sequence of thedrug-metabolizing enzyme peptide. For example, one class ofsubstitutions are conserved amino acid substitution. Such substitutionsare those that substitute a given amino acid in a drug-metabolizingenzyme peptide by another amino acid of like characteristics. Typicallyseen as conservative substitutions are the replacements, one foranother, among the aliphatic amino acids Ala, Val, Leu, and Ile;interchange of the hydroxyl residues Ser and Thr; exchange of the acidicresidues Asp and Glu; substitution between the amide residues Asn andGln; exchange of the basic residues Lys and Arg; and replacements amongthe aromatic residues Phe and Tyr. Guidance concerning which amino acidchanges are likely to be phenotypically silent are found in Bowie etal., Science 247:1306-1310 (1990).

[0061] Variant drug-metabolizing enzyme peptides can be fully functionalor can lack function in one or more activities, e.g. ability to bindsubstrate, ability to phosphorylate substrate, ability to mediatesignaling, etc. Fully functional variants typically contain onlyconservative variation or variation in non-critical residues or innon-critical regions. FIG. 2 provides the result of protein analysis andcan be used to identify critical domains/regions. Functional variantscan also contain substitution of similar amino acids that result in nochange or an insignificant change in function. Alternatively, suchsubstitutions may positively or negatively affect function to somedegree.

[0062] Non-functional variants typically contain one or morenon-conservative amino acid substitutions, deletions, insertions,inversions, or truncation or a substitution, insertion, inversion, ordeletion in a critical residue or critical region.

[0063] Amino acids that are essential for function can be identified bymethods known in the art, such as site-directed mutagenesis oralanine-scanning mutagenesis (Cunningham et al., Science 244:1081-1085(1989)), particularly using the results provided in FIG. 2. The latterprocedure introduces single alanine mutations at every residue in themolecule. The resulting mutant molecules are then tested for biologicalactivity such as drug-metabolizing enzyme activity or in assays such asan in vitro proliferative activity. Sites that are critical for bindingpartner/substrate binding can also be determined by structural analysissuch as crystallization, nuclear magnetic resonance or photoaffinitylabeling (Smith et al., J. Mol. Biol. 224:899-904 (1992); de Vos et al.Science.255:306-312 (1992)).

[0064] The present invention further provides fragments of thedrug-metabolizing enzyme peptides, in addition to proteins and peptidesthat comprise and consist of such fragments, particularly thosecomprising the residues identified in FIG. 2. The fragments to which theinvention pertains, however, are not to be construed as encompassingfragments that may be disclosed publicly prior to the present invention.

[0065] As used herein, a fragment comprises at least 8, 10, 12, 14, 16,or more contiguous amino acid residues from a drug-metabolizing enzymepeptide. Such fragments can be chosen based on the ability to retain oneor more of the biological activities of the drug-metabolizing enzymepeptide or could be chosen for the ability to perform a function, e.g.bind a substrate or act as an immunogen. Particularly importantfragments are biologically active fragments, peptides that are, forexample, about 8 or more amino acids in length. Such fragments willtypically comprise a domain or motif of the drug-metabolizing enzymepeptide, e.g., active site, a transmembrane domain or asubstrate-binding domain. Further, possible fragments include, but arenot limited to, domain or motif containing fragments, soluble peptidefragments, and fragments containing immunogenic structures. Predicteddomains and functional sites are readily identifiable by computerprograms well known and readily available to those of skill in the art(e.g., PROSITE analysis). The results of one such analysis are providedin FIG. 2.

[0066] Polypeptides often contain amino acids other than the 20 aminoacids commonly referred to as the 20 naturally occurring amino acids.Further, many amino acids, including the terminal amino acids, may bemodified by natural processes, such as processing and otherpost-translational modifications, or by chemical modification techniqueswell known in the art. Common modifications that occur naturally indrug-metabolizing enzyme peptides are described in basic texts, detailedmonographs, and the research literature, and they are well known tothose of skill in the art (some of these features are identified in FIG.2).

[0067] Known modifications include, but are not limited to, acetylation,acylation, ADP-ribosylation, amidation, covalent attachment of flavin,covalent attachment of a heme moiety, covalent attachment of anucleotide or nucleotide derivative, covalent attachment of a lipid orlipid derivative, covalent attachment of phosphotidylinositol,cross-linking, cyclization, disulfide bond formation, demethylation,formation of covalent crosslinks, formation of cystine, formation ofpyroglutamate, formylation, gamma carboxylation, glycosylation, GPIanchor formation, hydroxylation, iodination, methylation,myristoylation, oxidation, proteolytic processing, phosphorylation,prenylation, racemization, selenoylation, sulfation, transfer-RNAmediated addition of amino acids to proteins such as arginylation, andubiquitination.

[0068] Such modifications are well known to those of skill in the artand have been described in great detail in the scientific literature.Several particularly common modifications, glycosylation, lipidattachment, sulfation, gamma-carboxylation of glutamic acid residues,hydroxylation and ADP-ribosylation, for instance, are described in mostbasic texts, such as Proteins—Structure and Molecular Properties, 2ndEd., T. E. Creighton, W. H. Freeman and Company, New York (1993). Manydetailed reviews are available on this subject, such as by Wold, F.,Posttranslational Covalent Modification of Proteins, B. C. Johnson, Ed.,Academic Press, New York 1-12 (1983); Seifter et al. (Meth. Enzymol.182: 626-646 (1990)) and Rattan et al. (Ann. N.Y. Acad. Sci. 663:48-62(1992)).

[0069] Accordingly, the drug-metabolizing enzyme peptides of the presentinvention also encompass derivatives or analogs in which a substitutedamino acid residue is not one encoded by the genetic code, in which asubstituent group is included, in which the mature drug-metabolizingenzyme peptide is fused with another compound, such as a compound toincrease the half-life of the drug-metabolizing enzyme peptide (forexample, polyethylene glycol), or in which the additional amino acidsare fused to the mature drug-metabolizing enzyme peptide, such as aleader or secretory sequence or a sequence for purification of themature drug-metabolizing enzyme peptide or a pro-protein sequence.

[0070] Protein/Peptide Uses

[0071] The proteins of the present invention can be used in substantialand specific assays related to the functional information provided inthe Figures; to raise antibodies or to elicit another immune response;as a reagent (including the labeled reagent) in assays designed toquantitatively determine levels of the protein (or its binding partneror ligand) in biological fluids; and as markers for tissues in which thecorresponding protein is preferentially expressed (either constitutivelyor at a particular stage of tissue differentiation or development or ina disease state). Where the protein binds or potentially binds toanother protein or ligand (such as, for example, in a drug-metabolizingenzyme-effector protein interaction or drug-metabolizing enzyme-ligandinteraction), the protein can be used to identify the bindingpartner/ligand so as to develop a system to identify inhibitors of thebinding interaction. Any or all of these uses are capable of beingdeveloped into reagent grade or kit format for commercialization ascommercial products.

[0072] Methods for performing the uses listed above are well known tothose skilled in the art. References disclosing such methods include“Molecular Cloning: A Laboratory Manual”, 2d ed., Cold Spring HarborLaboratory Press, Sambrook, J., E. F. Fritsch and T. Maniatis eds.,1989, and “Methods in Enzymology: Guide to Molecular CloningTechniques”, Academic Press, Berger, S. L. and A. R. Kimmel eds., 1987.

[0073] The potential uses of the peptides of the present invention arebased primarily on the source of the protein as well as the class/actionof the protein. For example, drug-metabolizing enzymes isolated fromhumans and their human/mammalian orthologs serve as targets foridentifying agents for use in mammalian therapeutic applications, e.g. ahuman drug, particularly in modulating a biological or pathologicalresponse in a cell or tissue that expresses the drug-metabolizingenzyme. Experimental data as provided in FIG. 1 indicates thatdrug-metabolizing proteins of the present invention are expressed inhumans in the eye, kidney, and hepatocellular carcinoma, as indicated byvirtual northern blot analysis. In addition, PCR-based tissue screeningpanels indicate expression in a mixedbrain/heart/kidney/lung/spleen/testis/leukocyte sample. A largepercentage of pharmaceutical agents are being developed that modulatethe activity of drug-metabolizing enzyme proteins, particularly membersof the UDP-glycosyltransferase subfamily (see Background of theInvention). The structural and functional information provided in theBackground and Figures provide specific and substantial uses for themolecules of the present invention, particularly in combination with theexpression information provided in FIG. 1. Experimental data as providedin FIG. 1 indicates expression in humans in the eye, kidney,hepatocellular carcinoma, and a mixedbrain/heart/kidney/lung/spleen/testis/leukocyte sample. Such uses canreadily be determined using the information provided herein, that whichis known in the art, and routine experimentation.

[0074] The drug-metabolizing enzyme polypeptides (including variants andfragments that may have been disclosed prior to the present invention)are useful for biological assays related to drug-metabolizing enzymesthat are related to members of the UDP-glycosyltransferase subfamily.Such assays involve any of the known drug-metabolizing enzyme functionsor activities or properties useful for diagnosis and treatment ofdrug-metabolizing enzyme-related conditions that are specific for thesubfamily of drug-metabolizing enzymes that the one of the presentinvention belongs to, particularly in cells and tissues that express thedrug-metabolizing enzyme. Experimental data as provided in FIG. 1indicates that drug-metabolizing proteins of the present invention areexpressed in humans in the eye, kidney, and hepatocellular carcinoma, asindicated by virtual northern blot analysis. In addition, PCR-basedtissue screening panels indicate expression in a mixedbrain/heart/kidney/lung/spleen/testis/leukocyte sample.

[0075] The drug-metabolizing enzyme polypeptides are also useful in drugscreening assays, in cell-based or cell-free systems. Cell-based systemscan be native, i.e., cells that normally express the drug-metabolizingenzyme, as a biopsy or expanded in cell culture. Experimental data asprovided in FIG. 1 indicates expression in humans in the eye, kidney,hepatocellular carcinoma, and a mixedbrain/heart/kidney/lung/spleen/testis/leukocyte sample. In an alternateembodiment, cell-based assays involve recombinant host cells expressingthe drug-metabolizing enzyme protein.

[0076] The polypeptides can be used to identify compounds that modulatedrug-metabolizing enzyme activity of the protein in its natural state oran altered form that causes a specific disease or pathology associatedwith the drug-metabolizing enzyme. Both the drug-metabolizing enzymes ofthe present invention and appropriate variants and fragments can be usedin high-throughput screens to assay candidate compounds for the abilityto bind to the drug-metabolizing enzyme. These compounds can be furtherscreened against a functional drug-metabolizing enzyme to determine theeffect of the compound on the drug-metabolizing enzyme activity.Further, these compounds can be tested in animal or invertebrate systemsto determine activity/effectiveness. Compounds can be identified thatactivate (agonist) or inactivate (antagonist) the drug-metabolizingenzyme to a desired degree.

[0077] Further, the drug-metabolizing enzyme polypeptides can be used toscreen a compound for the ability to stimulate or inhibit interactionbetween the drug-metabolizing enzyme protein and a molecule thatnormally interacts with the drug-metabolizing enzyme protein. Suchassays typically include the steps of combining the drug-metabolizingenzyme protein with a candidate compound under conditions that allow thedrug-metabolizing enzyme protein, or fragment, to interact with thetarget molecule, and to detect the formation of a complex between theprotein and the target or to detect the biochemical consequence of theinteraction with the drug-metabolizing enzyme protein and the target.

[0078] Candidate compounds include, for example, 1) peptides such assoluble peptides, including Ig-tailed fusion peptides and members ofrandom peptide libraries (see, e.g., Lam et al., Nature 354:82-84(1991); Houghten et al., Nature 354:84-86 (1991)) and combinatorialchemistry-derived molecular libraries made of D- and/or L-configurationamino acids; 2) phosphopeptides (e.g., members of random and partiallydegenerate, directed phosphopeptide libraries, see, e.g., Songyang etal., Cell 72:767-778 (1993)); 3) antibodies (e.g., polyclonal,monoclonal, humanized, anti-idiotypic, chimeric, and single chainantibodies as well as Fab, F(ab′)₂, Fab expression library fragments,and epitope-binding fragments of antibodies); and 4) small organic andinorganic molecules (e.g., molecules obtained from combinatorial andnatural product libraries).

[0079] One candidate compound is a soluble fragment of the receptor thatcompetes for substrate binding. Other candidate compounds include mutantdrug-metabolizing enzymes or appropriate fragments containing mutationsthat affect drug-metabolizing enzyme function and thus compete forsubstrate. Accordingly, a fragment that competes for substrate, forexample with a higher affinity, or a fragment that binds substrate butdoes not allow release, is encompassed by the invention.

[0080] Any of the biological or biochemical functions mediated by thedrug-metabolizing enzyme can be used as an endpoint assay. These includeall of the biochemical or biochemical/biological events describedherein, in the references cited herein, incorporated by reference forthese endpoint assay targets, and other functions known to those ofordinary skill in the art or that can be readily identified using theinformation provided in the Figures, particularly FIG. 2. Specifically,a biological function of a cell or tissues that expresses thedrug-metabolizing enzyme can be assayed. Experimental data as providedin FIG. 1 indicates that drug-metabolizing proteins of the presentinvention are expressed in humans in the eye, kidney, and hepatocellularcarcinoma, as indicated by virtual northern blot analysis. In addition,PCR-based tissue screening panels indicate expression in a mixedbrain/heart/kidney/lung/spleen/testis/leukocyte sample.

[0081] Binding and/or activating compounds can also be screened by usingchimeric drug-metabolizing enzyme proteins in which the amino terminalextracellular domain, or parts thereof, the entire transmembrane domainor subregions, such as any of the seven transmembrane segments or any ofthe intracellular or extracellular loops and the carboxy terminalintracellular domain, or parts thereof, can be replaced by heterologousdomains or subregions. For example, a substrate-binding region can beused that interacts with a different substrate then that which isrecognized by the native drug-metabolizing enzyme. Accordingly, adifferent set of signal transduction components is available as anend-point assay for activation. This allows for assays to be performedin other than the specific host cell from which the drug-metabolizingenzyme is derived.

[0082] The drug-metabolizing enzyme polypeptides are also useful incompetition binding assays in methods designed to discover compoundsthat interact with the drug-metabolizing enzyme (e.g. binding partnersand/or ligands). Thus, a compound is exposed to a drug-metabolizingenzyme polypeptide under conditions that allow the compound to bind orto otherwise interact with the polypeptide. Soluble drug-metabolizingenzyme polypeptide is also added to the mixture. If the test compoundinteracts with the soluble drug-metabolizing enzyme polypeptide, itdecreases the amount of complex formed or activity from thedrug-metabolizing enzyme target. This type of assay is particularlyuseful in cases in which compounds are sought that interact withspecific regions of the drug-metabolizing enzyme. Thus, the solublepolypeptide that competes with the target drug-metabolizing enzymeregion is designed to contain peptide sequences corresponding to theregion of interest.

[0083] To perform cell free drug screening assays, it is sometimesdesirable to immobilize either the drug-metabolizing enzyme protein, orfragment, or its target molecule to facilitate separation of complexesfrom uncomplexed forms of one or both of the proteins, as well as toaccommodate automation of the assay.

[0084] Techniques for immobilizing proteins on matrices can be used inthe drug screening assays. In one embodiment, a fusion protein can beprovided which adds a domain that allows the protein to be bound to amatrix. For example, glutathione-S-transferase fusion proteins can beadsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis,Mo.) or glutathione derivatized microtitre plates, which are thencombined with the cell lysates (e.g., ³⁵S-labeled) and the candidatecompound, and the mixture incubated under conditions conducive tocomplex formation (e.g., at physiological conditions for salt and pH).Following incubation, the beads are washed to remove any unbound label,and the matrix immobilized and radiolabel determined directly, or in thesupernatant after the complexes are dissociated. Alternatively, thecomplexes can be dissociated from the matrix, separated by SDS-PAGE, andthe level of drug-metabolizing enzyme-binding protein found in the beadfraction quantitated from the gel using standard electrophoretictechniques. For example, either the polypeptide or its target moleculecan be immobilized utilizing conjugation of biotin and streptavidinusing techniques well known in the art. Alternatively, antibodiesreactive with the protein but which do not interfere with binding of theprotein to its target molecule can be derivatized to the wells of theplate, and the protein trapped in the wells by antibody conjugation.Preparations of a drug-metabolizing enzyme-binding protein and acandidate compound are incubated in the drug-metabolizing enzymeprotein-presenting wells and the amount of complex trapped in the wellcan be quantitated. Methods for detecting such complexes, in addition tothose described above for the GST-immobilized complexes, includeimmunodetection of complexes using antibodies reactive with thedrug-metabolizing enzyme protein target molecule, or which are reactivewith drug-metabolizing enzyme protein and compete with the targetmolecule, as well as enzyme-linked assays which rely on detecting anenzymatic activity associated with the target molecule.

[0085] Agents that modulate one of the drug-metabolizing enzymes of thepresent invention can be identified using one or more of the aboveassays, alone or in combination. It is generally preferable to use acell-based or cell free system first and then confirm activity in ananimal or other model system. Such model systems are well known in theart and can readily be employed in this context.

[0086] Modulators of drug-metabolizing enzyme protein activityidentified according to these drug screening assays can be used to treata subject with a disorder mediated by the drug-metabolizing enzymepathway, by treating cells or tissues that express the drug-metabolizingenzyme. Experimental data as provided in FIG. 1 indicates expression inhumans in the eye, kidney, hepatocellular carcinoma, and a mixedbrain/heart/kidney/lung/spleen/testis/leukocyte sample. These methods oftreatment include the steps of administering a modulator ofdrug-metabolizing enzyme activity in a pharmaceutical composition to asubject in need of such treatment, the modulator being identified asdescribed herein.

[0087] In yet another aspect of the invention, the drug-metabolizingenzyme proteins can be used as “bait proteins” in a two-hybrid assay orthree-hybrid assay (see, e.g., U.S. Pat. No. 5,283,317; Zervos et al.(1993) Cell 72:223-232; Madura et al. (1993) J. Biol. Chem.268:12046-12054; Bartel et al. (1993) Biotechniques 14:920-924; Iwabuchiet al. (1993) Oncogene 8:1693-1696; and Brent WO94/10300), to identifyother proteins, which bind to or interact with the drug-metabolizingenzyme and are involved in drug-metabolizing enzyme activity. Suchdrug-metabolizing enzyme-binding proteins are likely to bedrug-metabolizing enzyme inhibitors.

[0088] The two-hybrid system is based on the modular nature of mosttranscription factors, which consist of separable DNA-binding andactivation domains. Briefly, the assay utilizes two different DNAconstructs. In one construct, the gene that codes for adrug-metabolizing enzyme protein is fused to a gene encoding the DNAbinding domain of a known transcription factor (e.g., GAL-4). In theother construct, a DNA sequence, from a library of DNA sequences, thatencodes an unidentified protein (“prey” or “sample”) is fused to a genethat codes for the activation domain of the known transcription factor.If the “bait” and the “prey” proteins are able to interact, in vivo,forming a drug-metabolizing enzyme-dependent complex, the DNA-bindingand activation domains of the transcription factor are brought intoclose proximity. This proximity allows transcription of a reporter gene(e.g., LacZ) which is operably linked to a transcriptional regulatorysite responsive to the transcription factor. Expression of the reportergene can be detected and cell colonies containing the functionaltranscription factor can be isolated and used to obtain the cloned genewhich encodes the protein which interacts with the drug-metabolizingenzyme protein.

[0089] This invention further pertains to novel agents identified by theabove-described screening assays. Accordingly, it is within the scope ofthis invention to further use an agent identified as described herein inan appropriate animal model. For example, an agent identified asdescribed herein (e.g., a drug-metabolizing enzyme-modulating agent, anantisense drug-metabolizing enzyme nucleic acid molecule, adrug-metabolizing enzyme-specific antibody, or a drug-metabolizingenzyme-binding partner) can be used in an animal or other model todetermine the efficacy, toxicity, or side effects of treatment with suchan agent. Alternatively, an agent identified as described herein can beused in an animal or other model to determine the mechanism of action ofsuch an agent. Furthermore, this invention pertains to uses of novelagents identified by the above-described screening assays for treatmentsas described herein.

[0090] The drug-metabolizing enzyme proteins of the present inventionare also useful to provide a target for diagnosing a disease orpredisposition to disease mediated by the peptide. Accordingly, theinvention provides methods for detecting the presence, or levels of, theprotein (or encoding mRNA) in a cell, tissue, or organism. Experimentaldata as provided in FIG. 1 indicates expression in humans in the eye,kidney, hepatocellular carcinoma, and a mixedbrain/heart/kidney/lung/spleen/testis/leukocyte sample. The methodinvolves contacting a biological sample with a compound capable ofinteracting with the drug-metabolizing enzyme protein such that theinteraction can be detected. Such an assay can be provided in a singledetection format or a multi-detection format such as an antibody chiparray.

[0091] One agent for detecting a protein in a sample is an antibodycapable of selectively binding to protein. A biological sample includestissues, cells and biological fluids isolated from a subject, as well astissues, cells and fluids present within a subject.

[0092] The peptides of the present invention also provide targets fordiagnosing active protein activity, disease, or predisposition todisease, in a patient having a variant peptide, particularly activitiesand conditions that are known for other members of the family ofproteins to which the present one belongs. Thus, the peptide can beisolated from a biological sample and assayed for the presence of agenetic mutation that results in aberrant peptide. This includes aminoacid substitution, deletion, insertion, rearrangement, (as the result ofaberrant splicing events), and inappropriate post-translationalmodification. Analytic methods include altered electrophoretic mobility,altered tryptic peptide digest, altered drug-metabolizing enzymeactivity in cell-based or cell-free assay, alteration in substrate orantibody-binding pattern, altered isoelectric point, direct amino acidsequencing, and any other of the known assay techniques useful fordetecting mutations in a protein. Such an assay can be provided in asingle detection format or a multi-detection format such as an antibodychip array.

[0093] In vitro techniques for detection of peptide include enzymelinked immunosorbent assays (ELISAs), Western blots,immunoprecipitations and immunofluorescence using a detection reagent,such as an antibody or protein binding agent. Alternatively, the peptidecan be detected in vivo in a subject by introducing into the subject alabeled anti-peptide antibody or other types of detection agent. Forexample, the antibody can be labeled with a radioactive marker whosepresence and location in a subject can be detected by standard imagingtechniques. Particularly useful are methods that detect the allelicvariant of a peptide expressed in a subject and methods which detectfragments of a peptide in a sample.

[0094] The peptides are also useful in pharmacogenomic analysis.Pharmacogenomics deal with clinically significant hereditary variationsin the response to drugs due to altered drug disposition and abnormalaction in affected persons. See, e.g., Eichelbaum, M. (Clin. Exp.Pharmacol. Physiol. 23(10-11):983-985 (1996)), and Linder, M. W. (Clin.Chem. 43(2):254-266 (1997)). The clinical outcomes of these variationsresult in severe toxicity of therapeutic drugs in certain individuals ortherapeutic failure of drugs in certain individuals as a result ofindividual variation in metabolism. Thus, the genotype of the individualcan determine the way a therapeutic compound acts on the body or the waythe body metabolizes the compound. Further, the activity of drugmetabolizing enzymes effects both the intensity and duration of drugaction. Thus, the pharmacogenomics of the individual permit theselection of effective compounds and effective dosages of such compoundsfor prophylactic or therapeutic treatment based on the individual'sgenotype. The discovery of genetic polymorphisms in some drugmetabolizing enzymes has explained why some patients do not obtain theexpected drug effects, show an exaggerated drug effect, or experienceserious toxicity from standard drug dosages. Polymorphisms can beexpressed in the phenotype of the extensive metabolizer and thephenotype of the poor metabolizer. Accordingly, genetic polymorphism maylead to allelic protein variants of the drug-metabolizing enzyme proteinin which one or more of the drug-metabolizing enzyme functions in onepopulation is different from those in another population. The peptidesthus allow a target to ascertain a genetic predisposition that canaffect treatment modality. Thus, in a ligand-based treatment,polymorphism may give rise to amino terminal extracellular domainsand/or other substrate-binding regions that are more or less active insubstrate binding, and drug-metabolizing enzyme activation. Accordingly,substrate dosage would necessarily be modified to maximize thetherapeutic effect within a given population containing a polymorphism.As an alternative to genotyping, specific polymorphic peptides could beidentified.

[0095] The peptides are also useful for treating a disordercharacterized by an absence of, inappropriate, or unwanted expression ofthe protein. Experimental data as provided in FIG. 1 indicatesexpression in humans in the eye, kidney, hepatocellular carcinoma, and amixed brain/heart/kidney/lung/spleen/testis/leukocyte sample.Accordingly, methods for treatment include the use of thedrug-metabolizing enzyme protein or fragments.

[0096] Antibodies

[0097] The invention also provides antibodies that selectively bind toone of the peptides of the present invention, a protein comprising sucha peptide, as well as variants and fragments thereof. As used herein, anantibody selectively binds a target peptide when it binds the targetpeptide and does not significantly bind to unrelated proteins. Anantibody is still considered to selectively bind a peptide even if italso binds to other proteins that are not substantially homologous withthe target peptide so long as such proteins share homology with afragment or domain of the peptide target of the antibody. In this case,it would be understood that antibody binding to the peptide is stillselective despite some degree of cross-reactivity.

[0098] As used herein, an antibody is defined in terms consistent withthat recognized within the art: they are multi-subunit proteins producedby a mammalian organism in response to an antigen challenge. Theantibodies of the present invention include polyclonal antibodies andmonoclonal antibodies, as well as fragments of such antibodies,including, but not limited to, Fab or F(ab′)₂, and Fv fragments.

[0099] Many methods are known for generating and/or identifyingantibodies to a given target peptide. Several such methods are describedby Harlow, Antibodies, Cold Spring Harbor Press, (1989).

[0100] In general, to generate antibodies, an isolated peptide is usedas an immunogen and is administered to a mammalian organism, such as arat, rabbit or mouse. The full-length protein, an antigenic peptidefragment or a fusion protein can be used. Particularly importantfragments are those covering functional domains, such as the domainsidentified in FIG. 2, and domain of sequence homology or divergenceamongst the family, such as those that can readily be identified usingprotein alignment methods and as presented in the Figures.

[0101] Antibodies are preferably prepared from regions or discretefragments of the drug-metabolizing enzyme proteins. Antibodies can beprepared from any region of the peptide as described herein. However,preferred regions will include those involved in function/activityand/or drug-metabolizing enzyme/binding partner interaction. FIG. 2 canbe used to identify particularly important regions while sequencealignment can be used to identify conserved and unique sequencefragments.

[0102] An antigenic fragment will typically comprise at least 8contiguous amino acid residues. The antigenic peptide can comprise,however, at least 10, 12, 14, 16 or more amino acid residues. Suchfragments can be selected on a physical property, such as fragmentscorrespond to regions that are located on the surface of the protein,e.g., hydrophilic regions or can be selected based on sequenceuniqueness (see FIG. 2).

[0103] Detection on an antibody of the present invention can befacilitated by coupling (i.e., physically linking) the antibody to adetectable substance. Examples of detectable substances include variousenzymes, prosthetic groups, fluorescent materials, luminescentmaterials, bioluminescent materials, and radioactive materials. Examplesof suitable enzymes include horseradish peroxidase, alkalinephosphatase, β-galactosidase, or acetylcholinesterase; examples ofsuitable prosthetic group complexes include streptavidin/biotin andavidin/biotin; examples of suitable fluorescent materials includeumbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; anexample of a luminescent material includes luminol; examples ofbioluminescent materials include luciferase, luciferin, and aequorin,and examples of suitable radioactive material include ¹²⁵I, ¹³¹I, ³⁵S or³H.

[0104] Antibody Uses

[0105] The antibodies can be used to isolate one of the proteins of thepresent invention by standard techniques, such as affinitychromatography or immunoprecipitation. The antibodies can facilitate thepurification of the natural protein from cells and recombinantlyproduced protein expressed in host cells. In addition, such antibodiesare useful to detect the presence of one of the proteins of the presentinvention in cells or tissues to determine the pattern of expression ofthe protein among various tissues in an organism and over the course ofnormal development. Experimental data as provided in FIG. 1 indicatesthat drug-metabolizing proteins of the present invention are expressedin humans in the eye, kidney, and hepatocellular carcinoma, as indicatedby virtual northern blot analysis. In addition, PCR-based tissuescreening panels indicate expression in a mixedbrain/heart/kidney/lung/spleen/testis/leukocyte sample. Further, suchantibodies can be used to detect protein in situ, in vitro, or in a celllysate or supernatant in order to evaluate the abundance and pattern ofexpression. Also, such antibodies can be used to assess abnormal tissuedistribution or abnormal expression during development or progression ofa biological condition. Antibody detection of circulating fragments ofthe full length protein can be used to identify turnover.

[0106] Further, the antibodies can be used to assess expression indisease states such as in active stages of the disease or in anindividual with a predisposition toward disease related to the protein'sfunction. When a disorder is caused by an inappropriate tissuedistribution, developmental expression, level of expression of theprotein, or expressed/processed form, the antibody can be preparedagainst the normal protein. Experimental data as provided in FIG. 1indicates: expression in humans in the eye, kidney, hepatocellularcarcinoma, and a mixed brain/heart/kidney/lung/spleen/testis/leukocytesample. If a disorder is characterized by a specific mutation in theprotein, antibodies specific for this mutant protein can be used toassay for the presence of the specific mutant protein.

[0107] The antibodies can also be used to assess normal and aberrantsubcellular localization of cells in the various tissues in an organism.Experimental data as provided in FIG. 1 indicates expression in humansin the eye, kidney, hepatocellular carcinoma, and a mixedbrain/heart/kidney/lung/spleen/testis/leukocyte sample. The diagnosticuses can be applied, not only in genetic testing, but also in monitoringa treatment modality. Accordingly, where treatment is ultimately aimedat correcting expression level or the presence of aberrant sequence andaberrant tissue distribution or developmental expression, antibodiesdirected against the protein or relevant fragments can be used tomonitor therapeutic efficacy.

[0108] Additionally, antibodies are useful in pharmacogenomic analysis.Thus, antibodies prepared against polymorphic proteins can be used toidentify individuals that require modified treatment modalities. Theantibodies are also useful as diagnostic tools as an immunologicalmarker for aberrant protein analyzed by electrophoretic mobility,isoelectric point, tryptic peptide digest, and other physical assaysknown to those in the art.

[0109] The antibodies are also useful for tissue typing. Experimentaldata as provided in FIG. 1 indicates expression in humans in the eye,kidney, hepatocellular carcinoma, and a mixedbrain/heart/kidney/lung/spleen/testis/leukocyte sample. Thus, where aspecific protein has been correlated with expression in a specifictissue, antibodies that are specific for this protein can be used toidentify a tissue type.

[0110] The antibodies are also useful for inhibiting protein function,for example, blocking the binding of the drug-metabolizing enzymepeptide to a binding partner such as a substrate. These uses can also beapplied in a therapeutic context in which treatment involves inhibitingthe protein's function. An antibody can be used, for example, to blockbinding, thus modulating (agonizing or antagonizing) the peptidesactivity. Antibodies can be prepared against specific fragmentscontaining sites required for function or against intact protein that isassociated with a cell or cell membrane. See FIG. 2 for structuralinformation relating to the proteins of the present invention.

[0111] The invention also encompasses kits for using antibodies todetect the presence of a protein in a biological sample. The kit cancomprise antibodies such as a labeled or labelable antibody and acompound or agent for detecting protein in a biological sample; meansfor determining the amount of protein in the sample; means for comparingthe amount of protein in the sample with a standard; and instructionsfor use. Such a kit can be supplied to detect a single protein orepitope or can be configured to detect one of a multitude of epitopes,such as in an antibody detection array. Arrays are described in detailbelow for nucleic acid arrays and similar methods have been developedfor antibody arrays.

[0112] Nucleic Acid Molecules

[0113] The present invention further provides isolated nucleic acidmolecules that encode a drug-metabolizing enzyme peptide or protein ofthe present invention (cDNA/transcript sequence provided in FIG. 1).Such nucleic acid molecules will consist of, consist essentially of, orcomprise a nucleotide sequence that encodes one of the drug-metabolizingenzyme peptides of the present invention, an allelic variant thereof, oran ortholog or paralog thereof.

[0114] As used herein, an “isolated” nucleic acid molecule is one thatis separated from other nucleic acid present in the natural source ofthe nucleic acid. Preferably, an “isolated” nucleic acid is free ofsequences that naturally flank the nucleic acid (i.e., sequences locatedat the 5′ and 3′ ends of the nucleic acid) in the genomic DNA of theorganism from which the nucleic acid is derived. However, there can besome flanking nucleotide sequences, for example up to about 5 KB, 4 KB,3 KB, 2 KB, or 1 KB or less, particularly contiguous peptide encodingsequences and peptide encoding sequences within the same gene butseparated by introns in the genomic sequence. The important point isthat the nucleic acid is isolated from remote and unimportant flankingsequences such that it can be subjected to the specific manipulationsdescribed herein such as recombinant expression, preparation of probesand primers, and other uses specific to the nucleic acid sequences.

[0115] Moreover, an “isolated” nucleic acid molecule, such as atranscript/cDNA molecule, can be substantially free of other cellularmaterial, or culture medium when produced by recombinant techniques, orchemical precursors or other chemicals when chemically synthesized.However, the nucleic acid molecule can be fused to other coding orregulatory sequences and still be considered isolated.

[0116] For example, recombinant DNA molecules contained in a vector areconsidered isolated. Further examples of isolated DNA molecules includerecombinant DNA molecules maintained in heterologous host cells orpurified (partially or substantially) DNA molecules in solution.Isolated RNA molecules include in vivo or in vitro RNA transcripts ofthe isolated DNA molecules of the present invention. Isolated nucleicacid molecules according to the present invention further include suchmolecules produced synthetically.

[0117] Accordingly, the present invention provides nucleic acidmolecules that consist of the nucleotide sequence shown in FIG. 1 (SEQID NO:1, cDNA/transcript sequence), or any nucleic acid molecule thatencodes the protein provided in FIG. 2, SEQ ID NO:2. A nucleic acidmolecule consists of a nucleotide sequence when the nucleotide sequenceis the complete nucleotide sequence of the nucleic acid molecule.

[0118] The present invention further provides nucleic acid moleculesthat consist essentially of the nucleotide sequence shown in FIG. 1 (SEQID NO:1, cDNA/transcript sequence), or any nucleic acid molecule thatencodes the protein provided in FIG. 2, SEQ ID NO:2. A nucleic acidmolecule consists essentially of a nucleotide sequence when such anucleotide sequence is present with only a few additional nucleic acidresidues in the final nucleic acid molecule.

[0119] The present invention further provides nucleic acid moleculesthat comprise the nucleotide sequences shown in FIG. 1 (SEQ ID NO:1,cDNA/transcript sequence), or any nucleic acid molecule that encodes theprotein provided in FIG. 2, SEQ ID NO:2. A nucleic acid moleculecomprises a nucleotide sequence when the nucleotide sequence is at leastpart of the final nucleotide sequence of the nucleic acid molecule. Insuch a fashion, the nucleic acid molecule can be only the nucleotidesequence or have additional nucleic acid residues, such as nucleic acidresidues that are naturally associated with it or heterologousnucleotide sequences. Such a nucleic acid molecule can have a fewadditional nucleotides or can comprises several hundred or moreadditional nucleotides. A brief description of how various types ofthese nucleic acid molecules can be readily made/isolated is providedbelow.

[0120] In FIG. 1, both coding and non-coding sequences are provided. Thenucleic acid molecules in the Figures may contain 5′ and 3′ non-codingsequences, unspliced intronic sequences, and gene regulatory regions. Ingeneral such sequence features are either noted in FIGS. 1 or canreadily be identified using computational tools known in the art. Asdiscussed below, some of the non-coding regions, particularly generegulatory elements such as promoters, are useful for a variety ofpurposes, e.g. control of heterologous gene expression, target foridentifying gene activity modulating compounds, and are particularlyclaimed as fragments of the nucleic acid sequence provided herein.

[0121] The isolated nucleic acid molecules can encode the mature proteinplus additional amino or carboxyl-terminal amino acids, or amino acidsinterior to the mature peptide (when the mature form has more than onepeptide chain, for instance). Such sequences may play a role inprocessing of a protein from precursor to a mature form, facilitateprotein trafficking, prolong or shorten protein half-life or facilitatemanipulation of a protein for assay or production, among other things.As generally is the case in situ, the additional amino acids may beprocessed away from the mature protein by cellular enzymes.

[0122] As mentioned above, the isolated nucleic acid molecules include,but are not limited to, the sequence encoding the drug-metabolizingenzyme peptide alone, the sequence encoding the mature peptide andadditional coding sequences, such as a leader or secretory sequence(e.g., a pre-pro or pro-protein sequence), the sequence encoding themature peptide, with or without the additional coding sequences, plusadditional non-coding sequences, for example introns and non-coding 5′and 3′ sequences such as transcribed but non-translated sequences thatplay a role in transcription, mRNA processing (including splicing andpolyadenylation signals), ribosome binding and stability of mRNA. Inaddition, the nucleic acid molecule may be fused to a marker sequenceencoding, for example, a peptide that facilitates purification.

[0123] Isolated nucleic acid molecules can be in the form of RNA, suchas mRNA, or in the form DNA, including cDNA and genomic DNA obtained bycloning or produced by chemical synthetic techniques or by a combinationthereof. The nucleic acid, especially DNA, can be double-stranded orsingle-stranded. Single-stranded nucleic acid can be the coding strand(sense strand) or the non-coding strand (anti-sense strand).

[0124] The invention further provides nucleic acid molecules that encodefragments of the peptides of the present invention as well as nucleicacid molecules that encode obvious variants of the drug-metabolizingenzyme proteins of the present invention that are described above. Suchnucleic acid molecules may be naturally occurring, such as allelicvariants (same locus), paralogs (different locus), and orthologs(different organism), or may be constructed by recombinant DNA methodsor by chemical synthesis. Such non-naturally occurring variants may bemade by mutagenesis techniques, including those applied to nucleic acidmolecules, cells, or organisms. Accordingly, as discussed above, thevariants can contain nucleotide substitutions, deletions, inversions andinsertions. Variation can occur in either or both the coding andnon-coding regions. The variations can produce both conservative andnon-conservative amino acid substitutions.

[0125] The present invention further provides non-coding fragments ofthe nucleic acid molecules provided in FIG. 1. Preferred non-codingfragments include, but are not limited to, promoter sequences, enhancersequences, gene modulating sequences and gene termination sequences.Such fragments are useful in controlling heterologous gene expressionand in developing screens to identify gene-modulating agents. A promotercan readily be identified as being 5′ to the ATG start site in thecDNA/transcript sequence provided in FIG. 1.

[0126] A fragment comprises a contiguous nucleotide sequence greaterthan 12 or more nucleotides. Further, a fragment could at least 30, 40,50, 100, 250 or 500 nucleotides in length. The length of the fragmentwill be based on its intended use. For example, the fragment can encodeepitope bearing regions of the peptide, or can be useful as DNA probesand primers. Such fragments can be isolated using the known nucleotidesequence to synthesize an oligonucleotide probe. A labeled probe canthen be used to screen a cDNA library, genomic DNA library, or mRNA toisolate nucleic acid corresponding to the coding region. Further,primers can be used in PCR reactions to clone specific regions of gene.

[0127] A probe/primer typically comprises substantially a purifiedoligonucleotide or oligonucleotide pair. The oligonucleotide typicallycomprises a region of nucleotide sequence that hybridizes understringent conditions to at least about 12, 20, 25, 40, 50 or moreconsecutive nucleotides.

[0128] Orthologs, homologs, and allelic variants can be identified usingmethods well known in the art. As described in the Peptide Section,these variants comprise a nucleotide sequence encoding a peptide that istypically 60-70%, 70-80%, 80-90%, and more typically at least about90-95% or more homologous to the nucleotide sequence shown in the Figuresheets or a fragment of this sequence. Such nucleic acid molecules canreadily be identified as being able to hybridize under moderate tostringent conditions, to the nucleotide sequence shown in the Figuresheets or a fragment of the sequence. Allelic variants can readily bedetermined by genetic locus of the encoding gene.

[0129] As used herein, the term “hybridizes under stringent conditions”is intended to describe conditions for hybridization and washing underwhich nucleotide sequences encoding a peptide at least 60-70% homologousto each other typically remain hybridized to each other. The conditionscan be such that sequences at least about 60%, at least about 70%, or atleast about 80% or more homologous to each other typically remainhybridized to each other. Such stringent conditions are known to thoseskilled in the art and can be found in Current Protocols in MolecularBiology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. One example ofstringent hybridization conditions are hybridization in 6× sodiumchloride/sodium citrate (SSC) at about 45C, followed by one or morewashes in 0.2×SSC, 0.1% SDS at 50-65C. Examples of moderate to lowstringency hybridization conditions are well known in the art.

[0130] Nucleic Acid Molecule Uses

[0131] The nucleic acid molecules of the present invention are usefulfor probes, primers, chemical intermediates, and in biological assays.The nucleic acid molecules are useful as a hybridization probe formessenger RNA, transcript/cDNA and genomic DNA to isolate full-lengthcDNA and genomic clones encoding the peptide described in FIG. 2 and toisolate cDNA and genomic clones that correspond to variants (alleles,orthologs, etc.) producing the same or related peptides shown in FIG. 2.

[0132] The probe can correspond to any sequence along the entire lengthof the nucleic acid molecules provided in the Figures. Accordingly, itcould be derived from 5′ noncoding regions, the coding region, and 3′noncoding regions. However, as discussed, fragments are not to beconstrued as encompassing fragments disclosed prior to the presentinvention.

[0133] The nucleic acid molecules are also useful as primers for PCR toamplify any given region of a nucleic acid molecule and are useful tosynthesize antisense molecules of desired length and sequence.

[0134] The nucleic acid molecules are also useful for constructingrecombinant vectors. Such vectors include expression vectors thatexpress a portion of, or all of, the peptide sequences. Vectors alsoinclude insertion vectors, used to integrate into another nucleic acidmolecule sequence, such as into the cellular genome, to alter in situexpression of a gene and/or gene product. For example, an endogenouscoding sequence can be replaced via homologous recombination with all orpart of the coding region containing one or more specifically introducedmutations.

[0135] The nucleic acid molecules are also useful for expressingantigenic portions of the proteins.

[0136] The nucleic acid molecules are also useful as probes fordetermining the chromosomal positions of the nucleic acid molecules bymeans of in situ hybridization methods.

[0137] The nucleic acid molecules are also useful in making vectorscontaining the gene regulatory regions of the nucleic acid molecules ofthe present invention.

[0138] The nucleic acid molecules are also useful for designingribozymes corresponding to all, or a part, of the mRNA produced from thenucleic acid molecules described herein.

[0139] The nucleic acid molecules are also useful for making vectorsthat express part, or all, of the peptides.

[0140] The nucleic acid molecules are also useful for constructing hostcells expressing a part, or all, of the nucleic acid molecules andpeptides.

[0141] The nucleic acid molecules are also useful for constructingtransgenic animals expressing all, or a part, of the nucleic acidmolecules and peptides.

[0142] The nucleic acid molecules are also useful as hybridizationprobes for determining the presence, level, form and distribution ofnucleic acid expression. Experimental data as provided in FIG. 1indicates that drug-metabolizing proteins of the present invention areexpressed in humans in the eye, kidney, and hepatocellular carcinoma, asindicated by virtual northern blot analysis. In addition, PCR-basedtissue screening panels indicate expression in a mixedbrain/heart/kidney/lung/spleen/testis/leukocyte sample. Accordingly, theprobes can be used to detect the presence of, or to determine levels of,a specific nucleic acid molecule in cells, tissues, and in organisms.The nucleic acid whose level is determined can be DNA or RNA.Accordingly, probes corresponding to the peptides described herein canbe used to assess expression and/or gene copy number in a given cell,tissue, or organism. These uses are relevant for diagnosis of disordersinvolving an increase or decrease in drug-metabolizing enzyme proteinexpression relative to normal results.

[0143] In vitro techniques for detection of mRNA include Northernhybridizations and in situ hybridizations. In vitro techniques fordetecting DNA include Southern hybridizations and in situ hybridization.

[0144] Probes can be used as a part of a diagnostic test kit foridentifying cells or tissues that express a drug-metabolizing enzymeprotein, such as by measuring a level of a drug-metabolizingenzyme-encoding nucleic acid in a sample of cells from a subject e.g.,mRNA or genomic DNA, or determining if a drug-metabolizing enzyme genehas been mutated. Experimental data as provided in FIG. 1 indicates thatdrug-metabolizing proteins of the present invention are expressed inhumans in the eye, kidney, and hepatocellular carcinoma, as indicated byvirtual northern blot analysis. In addition, PCR-based tissue screeningpanels indicate expression in a mixedbrain/heart/kidney/lung/spleen/testis/leukocyte sample.

[0145] Nucleic acid expression assays are useful for drug screening toidentify compounds that modulate drug-metabolizing enzyme nucleic acidexpression.

[0146] The invention thus provides a method for identifying a compoundthat can be used to treat a disorder associated with nucleic acidexpression of the drug-metabolizing enzyme gene, particularly biologicaland pathological processes that are mediated by the drug-metabolizingenzyme in cells and tissues that express it. Experimental data asprovided in FIG. 1 indicates expression in humans in the eye, kidney,hepatocellular carcinoma, and a mixedbrain/heart/kidney/lung/spleen/testis/leukocyte sample. The methodtypically includes assaying the ability of the compound to modulate theexpression of the drug-metabolizing enzyme nucleic acid and thusidentifying a compound that can be used to treat a disordercharacterized by undesired drug-metabolizing enzyme nucleic acidexpression. The assays can be performed in cell-based and cell-freesystems. Cell-based assays include cells naturally expressing thedrug-metabolizing enzyme nucleic acid or recombinant cells geneticallyengineered to express specific nucleic acid sequences.

[0147] Thus, modulators of drug-metabolizing enzyme gene expression canbe identified in a method wherein a cell is contacted with a candidatecompound and the expression of mRNA determined. The level of expressionof drug-metabolizing enzyme mRNA in the presence of the candidatecompound is compared to the level of expression of drug-metabolizingenzyme mRNA in the absence of the candidate compound. The candidatecompound can then be identified as a modulator of nucleic acidexpression based on this comparison and be used, for example to treat adisorder characterized by aberrant nucleic acid expression. Whenexpression of mRNA is statistically significantly greater in thepresence of the candidate compound than in its absence, the candidatecompound is identified as a stimulator of nucleic acid expression. Whennucleic acid expression is statistically significantly less in thepresence of the candidate compound than in its absence, the candidatecompound is identified as an inhibitor of nucleic acid expression.

[0148] The invention further provides methods of treatment, with thenucleic acid as a target, using a compound identified through drugscreening as a gene modulator to modulate drug-metabolizing enzymenucleic acid expression in cells and tissues that express thedrug-metabolizing enzyme. Experimental data as provided in FIG. 1indicates that drug-metabolizing proteins of the present invention areexpressed in humans in the eye, kidney, and hepatocellular carcinoma, asindicated by virtual northern blot analysis. In addition, PCR-basedtissue screening panels indicate expression in a mixedbrain/heart/kidney/lung/spleen/testis/leukocyte sample. Modulationincludes both up-regulation (i.e. activation or agonization) ordown-regulation (suppression or antagonization) or nucleic acidexpression.

[0149] Alternatively, a modulator for drug-metabolizing enzyme nucleicacid expression can be a small molecule or drug identified using thescreening assays described herein as long as the drug or small moleculeinhibits the drug-metabolizing enzyme nucleic acid expression in thecells and tissues that express the protein. Experimental data asprovided in FIG. 1 indicates expression in humans in the eye, kidney,hepatocellular carcinoma, and a mixedbrain/heart/kidney/lung/spleen/testis/leukocyte sample.

[0150] The nucleic acid molecules are also useful for monitoring theeffectiveness of modulating compounds on the expression or activity ofthe drug-metabolizing enzyme gene in clinical trials or in a treatmentregimen. Thus, the gene expression pattern can serve as a barometer forthe continuing effectiveness of treatment with the compound,particularly with compounds to which a patient can develop resistance.The gene expression pattern can also serve as a marker indicative of aphysiological response of the affected cells to the compound.Accordingly, such monitoring would allow either increased administrationof the compound or the administration of alternative compounds to whichthe patient has not become resistant. Similarly, if the level of nucleicacid expression falls below a desirable level, administration of thecompound could be commensurately decreased.

[0151] The nucleic acid molecules are also useful in diagnostic assaysfor qualitative changes in drug-metabolizing enzyme nucleic acidexpression, and particularly in qualitative changes that lead topathology. The nucleic acid molecules can be used to detect mutations indrug-metabolizing enzyme genes and gene expression products such asmRNA. The nucleic acid molecules can be used as hybridization probes todetect naturally occurring genetic mutations in the drug-metabolizingenzyme gene and thereby to determine whether a subject with the mutationis at risk for a disorder caused by the mutation. Mutations includedeletion, addition, or substitution of one or more nucleotides in thegene, chromosomal rearrangement, such as inversion or transposition,modification of genomic DNA, such as aberrant methylation patterns orchanges in gene copy number, such as amplification. Detection of amutated form of the drug-metabolizing enzyme gene associated with adysfunction provides a diagnostic tool for an active disease orsusceptibility to disease when the disease results from overexpression,underexpression, or altered expression of a drug-metabolizing enzymeprotein.

[0152] Individuals carrying mutations in the drug-metabolizing enzymegene can be detected at the nucleic acid level by a variety oftechniques. Genomic DNA can be analyzed directly or can be amplified byusing PCR prior to analysis. RNA or cDNA can be used in the same way. Insome uses, detection of the mutation involves the use of a probe/primerin a polymerase chain reaction (PCR) (see, e.g. U.S. Pat. Nos. 4,683,195and 4,683,202), such as anchor PCR or RACE PCR, or, alternatively, in aligation chain reaction (LCR) (see, e.g., Landegran et al., Science241:1077-1080 (1988); and Nakazawa et al., PNAS 91:360-364 (1994)), thelatter of which can be particularly useful for detecting point mutationsin the gene (see Abravaya et al., Nucleic Acids Res. 23:675-682 (1995)).This method can include the steps of collecting a sample of cells from apatient, isolating nucleic acid (e.g., genomic, mRNA or both) from thecells of the sample, contacting the nucleic acid sample with one or moreprimers which specifically hybridize to a gene under conditions suchthat hybridization and amplification of the gene (if present) occurs,and detecting the presence or absence of an amplification product, ordetecting the size of the amplification product and comparing the lengthto a control sample. Deletions and insertions can be detected by achange in size of the amplified product compared to the normal genotype.Point mutations can be identified by hybridizing amplified DNA to normalRNA or antisense DNA sequences.

[0153] Alternatively, mutations in a drug-metabolizing enzyme gene canbe directly identified, for example, by alterations in restrictionenzyme digestion patterns determined by gel electrophoresis.

[0154] Further, sequence-specific ribozymes (U.S. Pat. No. 5,498,531)can be used to score for the presence of specific mutations bydevelopment or loss of a ribozyme cleavage site. Perfectly matchedsequences can be distinguished from mismatched sequences by nucleasecleavage digestion assays or by differences in melting temperature.

[0155] Sequence changes at specific locations can also be assessed bynuclease protection assays such as RNase and SI protection or thechemical cleavage method. Furthermore, sequence differences between amutant drug-metabolizing enzyme gene and a wild-type gene can bedetermined by direct DNA sequencing. A variety of automated sequencingprocedures can be utilized when performing the diagnostic assays (Naeve,C. W., (1995) Biotechniques 19:448), including sequencing by massspectrometry (see, e.g., PCT International Publication No. WO 94/16101;Cohen et al., Adv. Chromatogr. 36:127-162 (1996); and Griffin et al.,Appl. Biochem. Biotechnol. 38:147-159 (1993)).

[0156] Other methods for detecting mutations in the gene include methodsin which protection from cleavage agents is used to detect mismatchedbases in RNA/RNA or RNA/DNA duplexes (Myers et al., Science 230:1242(1985)); Cotton et al., PNAS 85:4397 (1988); Saleeba et al., Meth.Enzymol. 217:286-295 (1992)), electrophoretic mobility of mutant andwild type nucleic acid is compared (Orita et al., PNAS 86:2766 (1989);Cotton et al., Mutat. Res. 285:125-144 (1993); and Hayashi et al.,Genet. Anal. Tech. Appl. 9:73-79 (1992)), and movement of mutant orwild-type fragments in polyacrylamide gels containing a gradient ofdenaturant is assayed using denaturing gradient gel electrophoresis(Myers et al., Nature 313:495 (1985)). Examples of other techniques fordetecting point mutations include selective oligonucleotidehybridization, selective amplification, and selective primer extension.

[0157] The nucleic acid molecules are also useful for testing anindividual for a genotype that while not necessarily causing thedisease, nevertheless affects the treatment modality. Thus, the nucleicacid molecules can be used to study the relationship between anindividual's genotype and the individual's response to a compound usedfor treatment (pharmacogenomic relationship). Accordingly, the nucleicacid molecules described herein can be used to assess the mutationcontent of the drug-metabolizing enzyme gene in an individual in orderto select an appropriate compound or dosage regimen for treatment.

[0158] Thus nucleic acid molecules displaying genetic variations thataffect treatment provide a diagnostic target that can be used to tailortreatment in an individual. Accordingly, the production of recombinantcells and animals containing these polymorphisms allow effectiveclinical design of treatment compounds and dosage regimens.

[0159] The nucleic acid molecules are thus useful as antisenseconstructs to control drug-metabolizing enzyme gene expression in cells,tissues, and organisms. A DNA antisense nucleic acid molecule isdesigned to be complementary to a region of the gene involved intranscription, preventing transcription and hence production ofdrug-metabolizing enzyme protein. An antisense RNA or DNA nucleic acidmolecule would hybridize to the mRNA and thus block translation of mRNAinto drug-metabolizing enzyme protein.

[0160] Alternatively, a class of antisense molecules can be used toinactivate mRNA in order to decrease expression of drug-metabolizingenzyme nucleic acid. Accordingly, these molecules can treat a disordercharacterized by abnormal or undesired drug-metabolizing enzyme nucleicacid expression. This technique involves cleavage by means of ribozymescontaining nucleotide sequences complementary to one or more regions inthe mRNA that attenuate the ability of the mRNA to be translated.Possible regions include coding regions and particularly coding regionscorresponding to the catalytic and other functional activities of thedrug-metabolizing enzyme protein, such as substrate binding.

[0161] The nucleic acid molecules also provide vectors for gene therapyin patients containing cells that are aberrant in drug-metabolizingenzyme gene expression. Thus, recombinant cells, which include thepatient's cells that have been engineered ex vivo and returned to thepatient, are introduced into an individual where the cells produce thedesired drug-metabolizing enzyme protein to treat the individual.

[0162] The invention also encompasses kits for detecting the presence ofa drug-metabolizing enzyme nucleic acid in a biological sample.Experimental data as provided in FIG. 1 indicates that drug-metabolizingproteins of the present invention are expressed in humans in the eye,kidney, and hepatocellular carcinoma, as indicated by virtual northernblot analysis. In addition, PCR-based tissue screening panels indicateexpression in a mixed brain/heart/kidney/lung/spleen/testis/leukocytesample. For example, the kit can comprise reagents such as a labeled orlabelable nucleic acid or agent capable of detecting drug-metabolizingenzyme nucleic acid in a biological sample; means for determining theamount of drug-metabolizing enzyme nucleic acid in the sample; and meansfor comparing the amount of drug-metabolizing enzyme nucleic acid in thesample with a standard. The compound or agent can be packaged in asuitable container. The kit can further comprise instructions for usingthe kit to detect drug-metabolizing enzyme protein mRNA or DNA.

[0163] Nucleic Acid Arrays

[0164] The present invention further provides nucleic acid detectionkits, such as arrays or microarrays of nucleic acid molecules that arebased on the sequence information provided in FIGS. 1 (SEQ ID NO:1).

[0165] As used herein “Arrays” or “Microarrays” refers to an array ofdistinct polynucleotides or oligonucleotides synthesized on a substrate,such as paper, nylon or other type of membrane, filter, chip, glassslide, or any other suitable solid support. In one embodiment, themicroarray is prepared and used according to the methods described inU.S. Pat. No. 5,837,832, Chee et al., PCT application WO95/11995 (Cheeet al.), Lockhart, D. J. et al. (1996; Nat. Biotech. 14: 1675-1680) andSchena, M. et al. (1996; Proc. Natl. Acad. Sci. 93: 10614-10619), all ofwhich are incorporated herein in their entirety by reference. In otherembodiments, such arrays are produced by the methods described by Brownet al., U.S. Pat. No. 5,807,522.

[0166] The microarray or detection kit is preferably composed of a largenumber of unique, single-stranded nucleic acid sequences, usually eithersynthetic antisense oligonucleotides or fragments of cDNAs, fixed to asolid support. The oligonucleotides are preferably about 6-60nucleotides in length, more preferably 15-30 nucleotides in length, andmost preferably about 20-25 nucleotides in length. For a certain type ofmicroarray or detection kit, it may be preferable to useoligonucleotides that are only 7-20 nucleotides in length. Themicroarray or detection kit may contain oligonucleotides that cover theknown 5′, or 3′, sequence, sequential oligonucleotides that cover thefull length sequence; or unique oligonucleotides selected fromparticular areas along the length of the sequence. Polynucleotides usedin the microarray or detection kit may be oligonucleotides that arespecific to a gene or genes of interest.

[0167] In order to produce oligonucleotides to a known sequence for amicroarray or detection kit, the gene(s) of interest (or an ORFidentified from the contigs of the present invention) is typicallyexamined using a computer algorithm which starts at the 5′ or at the 3′end of the nucleotide sequence. Typical algorithms will then identifyoligomers of defined length that are unique to the gene, have a GCcontent within a range suitable for hybridization, and lack predictedsecondary structure that may interfere with hybridization. In certainsituations it may be appropriate to use pairs of oligonucleotides on amicroarray or detection kit. The “pairs” will be identical, except forone nucleotide that preferably is located in the center of the sequence.The second oligonucleotide in the pair (mismatched by one) serves as acontrol. The number of oligonucleotide pairs may range from two to onemillion. The oligomers are synthesized at designated areas on asubstrate using a light-directed chemical process. The substrate may bepaper, nylon or other type of membrane, filter, chip, glass slide or anyother suitable solid support.

[0168] In another aspect, an oligonucleotide may be synthesized on thesurface of the substrate by using a chemical coupling procedure and anink jet application apparatus, as described in PCT applicationWO95/251116 (Baldeschweiler et al.) which is incorporated herein in itsentirety by reference. In another aspect, a “gridded” array analogous toa dot (or slot) blot may be used to arrange and link cDNA fragments oroligonucleotides to the surface of a substrate using a vacuum system,thermal, V, mechanical or chemical bonding procedures. An array, such asthose described above, may be produced by hand or by using availabledevices (slot blot or dot blot apparatus), materials (any suitable solidsupport), and machines (including robotic instruments), and may contain8, 24, 96, 384, 1536, 6144 or more oligonucleotides, or any other numberbetween two and one million which lends itself to the efficient use ofcommercially available instrumentation.

[0169] In order to conduct sample analysis using a microarray ordetection kit, the RNA or DNA from a biological sample is made intohybridization probes. The mRNA is isolated, and cDNA is produced andused as a template to make antisense RNA (aRNA). The aRNA is amplifiedin the presence of fluorescent nucleotides, and labeled probes areincubated with the microarray or detection kit so that the probesequences hybridize to complementary oligonucleotides of the microarrayor detection kit. Incubation conditions are adjusted so thathybridization occurs with precise complementary matches or with variousdegrees of less complementarity. After removal of nonhybridized probes,a scanner is used to determine the levels and patterns of fluorescence.The scanned images are examined to determine degree of complementarityand the relative abundance of each oligonucleotide sequence on themicroarray or detection kit. The biological samples may be obtained fromany bodily fluids (such as blood, urine, saliva, phlegm, gastric juices,etc.), cultured cells, biopsies, or other tissue preparations. Adetection system may be used to measure the absence, presence, andamount of hybridization for all of the distinct sequencessimultaneously. This data may be used for large-scale correlationstudies on the sequences, expression patterns, mutations, variants, orpolymorphisms among samples.

[0170] Using such arrays, the present invention provides methods toidentify the expression of the drug-metabolizing enzymeproteins/peptides of the present invention. In detail, such methodscomprise incubating a test sample with one or more nucleic acidmolecules and assaying for binding of the nucleic acid molecule withcomponents within the test sample. Such assays will typically involvearrays comprising many genes, at least one of which is a gene of thepresent invention and or alleles of the drug-metabolizing enzyme gene ofthe present invention.

[0171] Conditions for incubating a nucleic acid molecule with a testsample vary. Incubation conditions depend on the format employed in theassay, the detection methods employed, and the type and nature of thenucleic acid molecule used in the assay. One skilled in the art willrecognize that any one of the commonly available hybridization,amplification or array assay formats can readily be adapted to employthe novel fragments of the Human genome disclosed herein. Examples ofsuch assays can be found in Chard, T, An Introduction toRadioimmunoassay and Related Techniques, Elsevier Science Publishers,Amsterdam, The Netherlands (1986); Bullock, G. R. et al., Techniques inImmunocytochemistry, Academic Press, Orlando, Fla. Vol. 1 (1982), Vol. 2(1983), Vol. 3 (1985); Tijssen, P., Practice and Theory of EnzymeImmunoassays: Laboratory Techniques in Biochemistry and MolecularBiology, Elsevier Science Publishers, Amsterdam, The Netherlands (1985).

[0172] The test samples of the present invention include cells, proteinor membrane extracts of cells. The test sample used in theabove-described method will vary based on the assay format, nature ofthe detection method and the tissues, cells or extracts used as thesample to be assayed. Methods for preparing nucleic acid extracts or ofcells are well known in the art and can be readily be adapted in orderto obtain a sample that is compatible with the system utilized.

[0173] In another embodiment of the present invention, kits are providedwhich contain the necessary reagents to carry out the assays of thepresent invention.

[0174] Specifically, the invention provides a compartmentalized kit toreceive, in close confinement, one or more containers which comprises:(a) a first container comprising one of the nucleic acid molecules thatcan bind to a fragment of the Human genome disclosed herein; and (b) oneor more other containers comprising one or more of the following: washreagents, reagents capable of detecting presence of a bound nucleicacid.

[0175] In detail, a compartmentalized kit includes any kit in whichreagents are contained in separate containers. Such containers includesmall glass containers, plastic containers, strips of plastic, glass orpaper, or arraying material such as silica. Such containers allows oneto efficiently transfer reagents from one compartment to anothercompartment such that the samples and reagents are notcross-contaminated, and the agents or solutions of each container can beadded in a quantitative fashion from one compartment to another. Suchcontainers will include a container which will accept the test sample, acontainer which contains the nucleic acid probe, containers whichcontain wash reagents (such as phosphate buffered saline, Tris-buffers,etc.), and containers which contain the reagents used to detect thebound probe. One skilled in the art will readily recognize that thepreviously unidentified drug-metabolizing enzyme gene of the presentinvention can be routinely identified using the sequence informationdisclosed herein can be readily incorporated into one of the establishedkit formats which are well known in the art, particularly expressionarrays.

[0176] Vectors/Host Cells

[0177] The invention also provides vectors containing the nucleic acidmolecules described herein. The term “vector” refers to a vehicle,preferably a nucleic acid molecule, which can transport the nucleic acidmolecules. When the vector is a nucleic acid molecule, the nucleic acidmolecules are covalently linked to the vector nucleic acid. With thisaspect of the invention, the vector includes a plasmid, single or doublestranded phage, a single or double stranded RNA or DNA viral vector, orartificial chromosome, such as a BAC, PAC, YAC, OR MAC.

[0178] A vector can be maintained in the host cell as anextrachromosomal element where it replicates and produces additionalcopies of the nucleic acid molecules. Alternatively, the vector mayintegrate into the host cell genome and produce additional copies of thenucleic acid molecules when the host cell replicates.

[0179] The invention provides Vectors for the maintenance (cloningvectors) or vectors for expression (expression vectors) of the nucleicacid molecules. The vectors can function in prokaryotic or eukaryoticcells or in both (shuttle vectors).

[0180] Expression vectors contain cis-acting regulatory regions that areoperably linked in the vector to the nucleic acid molecules such thattranscription of the nucleic acid molecules is allowed in a host cell.The nucleic acid molecules can be introduced into the host cell with aseparate nucleic acid molecule capable of affecting transcription. Thus,the second nucleic acid molecule may provide a trans-acting factorinteracting with the cis-regulatory control region to allowtranscription of the nucleic acid molecules from the vector.Alternatively, a trans-acting factor may be supplied by the host cell.Finally, a trans-acting factor can be produced from the vector itself.It is understood, however, that in some embodiments, transcriptionand/or translation of the nucleic acid molecules can occur in acell-free system.

[0181] The regulatory sequence to which the nucleic acid moleculesdescribed herein can be operably linked include promoters for directingmRNA transcription. These include, but are not limited to, the leftpromoter from bacteriophage λ, the lac, TRP, and TAC promoters from E.coli, the early and late promoters from SV40, the CMV immediate earlypromoter, the adenovirus early and late promoters, and retroviruslong-terminal repeats.

[0182] In addition to control regions that promote transcription,expression vectors may also include regions that modulate transcription,such as repressor binding sites and enhancers. Examples include the SV40enhancer, the cytomegalovirus immediate early enhancer, polyomaenhancer, adenovirus enhancers, and retrovirus LTR enhancers.

[0183] In addition to containing sites for transcription initiation andcontrol, expression vectors can also contain sequences necessary fortranscription termination and, in the transcribed region a ribosomebinding site for translation. Other regulatory control elements forexpression include initiation and termination codons as well aspolyadenylation signals. The person of ordinary skill in the art wouldbe aware of the numerous regulatory sequences that are useful inexpression vectors. Such regulatory sequences are described, forexample, in Sambrook et al., Molecular Cloning: A Laboratory Manual.2nd. ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,(1989).

[0184] A variety of expression vectors can be used to express a nucleicacid molecule. Such vectors include chromosomal, episomal, andvirus-derived vectors, for example vectors derived from bacterialplasmids, from bacteriophage, from yeast episomes, from yeastchromosomal elements, including yeast artificial chromosomes, fromviruses such as baculoviruses, papovaviruses such as SV40, Vacciniaviruses, adenoviruses, pox viruses, pseudorabies viruses, andretroviruses. Vectors may also be derived from combinations of thesesources such as those derived from plasmid and bacteriophage geneticelements, e.g. cosmids and phagemids. Appropriate cloning and expressionvectors for prokaryotic and eukaryotic hosts are described in Sambrooket al., Molecular Cloning: A Laboratory Manual. 2nd. ed., Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., (1989).

[0185] The regulatory sequence may provide constitutive expression inone or more host cells (i.e. tissue specific) or may provide forinducible expression in one or more cell types such as by temperature,nutrient additive, or exogenous factor such as a hormone or otherligand. A variety of vectors providing for constitutive and inducibleexpression in prokaryotic and eukaryotic hosts are well known to thoseof ordinary skill in the art.

[0186] The nucleic acid molecules can be inserted into the vectornucleic acid by well-known methodology. Generally, the DNA sequence thatwill ultimately be expressed is joined to an expression vector bycleaving the DNA sequence and the expression vector with one or morerestriction enzymes and then ligating the fragments together. Proceduresfor restriction enzyme digestion and ligation are well known to those ofordinary skill in the art.

[0187] The vector containing the appropriate nucleic acid molecule canbe introduced into an appropriate host cell for propagation orexpression using well-known techniques. Bacterial cells include, but arenot limited to, E. coli, Streptomyces, and Salmonella typhimurium.Eukaryotic cells include, but are not limited to, yeast, insect cellssuch as Drosophila, animal cells such as COS and CHO cells, and plantcells.

[0188] As described herein, it may be desirable to express the peptideas a fusion protein. Accordingly, the invention provides fusion vectorsthat allow for the production of the peptides. Fusion vectors canincrease the expression of a recombinant protein, increase thesolubility of the recombinant protein, and aid in the purification ofthe protein by acting for example as a ligand for affinity purification.A proteolytic cleavage site may be introduced at the junction of thefusion moiety so that the desired peptide can ultimately be separatedfrom the fusion moiety. Proteolytic enzymes include, but are not limitedto, factor Xa, thrombin, and enterokinase. Typical fusion expressionvectors include pGEX (Smith et al., Gene 67:31-40 (1988)), pMAL (NewEngland Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.)which fuse glutathione S-transferase (GST), maltose E binding protein,or protein A, respectively, to the target recombinant protein. Examplesof suitable inducible non-fusion E. coli expression vectors include pTrc(Amann et al., Gene 69:301-315 (1988)) and pET 11d (Studier et al., GeneExpression Technology: Methods in Enzymology 185:60-89 (1990)).

[0189] Recombinant protein expression can be maximized in host bacteriaby providing a genetic background wherein the host cell has an impairedcapacity to proteolytically cleave the recombinant protein. (Gottesman,S., Gene Expression Technology: Methods in Enzymology 185, AcademicPress, San Diego, Calif. (1990) 119-128). Alternatively, the sequence ofthe nucleic acid molecule of interest can be altered to providepreferential codon usage for a specific host cell, for example E. coli.(Wada et al., Nucleic Acids Res. 20:2111-2118 (1992)).

[0190] The nucleic acid molecules can also be expressed by expressionvectors that are operative in yeast. Examples of vectors for expressionin yeast e.g., S. cerevisiae include pYepSec1 (Baldari, et al., EMBO J.6:229-234 (1987)), pMFa (Kurjan et al., Cell 30:933-943(1982)), pJRY88(Schultz et al., Gene 54:113-123 (1987)), and pYES2 (InvitrogenCorporation, San Diego, Calif.).

[0191] The nucleic acid molecules can also be expressed in insect cellsusing, for example, baculovirus expression vectors. Baculovirus vectorsavailable for expression of proteins in cultured insect cells (e.g., Sf9 cells) include the pAc series (Smith et al., Mol. Cell Biol.3:2156-2165 (1983)) and the pVL series (Lucklow et al., Virology170:31-39 (1989)).

[0192] In certain embodiments of the invention, the nucleic acidmolecules described herein are expressed in mammalian cells usingmammalian expression vectors. Examples of mammalian expression vectorsinclude pCDM8 (Seed, B. Nature 329:840(1987)) and pMT2PC (Kaufman etal., EMBO J. 6:187-195 (1987)).

[0193] The expression vectors listed herein are provided by way ofexample only of the well-known vectors available to those of ordinaryskill in the art that would be useful to express the nucleic acidmolecules. The person of ordinary skill in the art would be aware ofother vectors suitable for maintenance propagation or expression of thenucleic acid molecules described herein. These are found for example inSambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: ALaboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.

[0194] The invention also encompasses vectors in which the nucleic acidsequences described herein are cloned into the vector in reverseorientation, but operably linked to a regulatory sequence that permitstranscription of antisense RNA. Thus, an antisense transcript can beproduced to all, or to a portion, of the nucleic acid molecule sequencesdescribed herein, including both coding and non-coding regions.Expression of this antisense RNA is subject to each of the parametersdescribed above in relation to expression of the sense RNA (regulatorysequences, constitutive or inducible expression, tissue-specificexpression).

[0195] The invention also relates to recombinant host cells containingthe vectors described herein. Host cells therefore include prokaryoticcells, lower eukaryotic cells such as yeast, other eukaryotic cells suchas insect cells, and higher eukaryotic cells such as mammalian cells.

[0196] The recombinant host cells are prepared by introducing the vectorconstructs described herein into the cells by techniques readilyavailable to the person of ordinary skill in the art. These include, butare not limited to, calcium phosphate transfection,DEAE-dextran-mediated transfection, cationic lipid-mediatedtransfection, electroporation, transduction, infection, lipofection, andother techniques such as those found in Sambrook, et al. (MolecularCloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989).

[0197] Host cells can contain more than one vector. Thus, differentnucleotide sequences can be introduced on different vectors of the samecell. Similarly, the nucleic acid molecules can be introduced eitheralone or with other nucleic acid molecules that are not related to thenucleic acid molecules such as those providing trans-acting factors forexpression vectors. When more than one vector is introduced into a cell,the vectors can be introduced independently, co-introduced or joined tothe nucleic acid molecule vector.

[0198] In the case of bacteriophage and viral vectors, these can beintroduced into cells as packaged or encapsulated virus by standardprocedures for infection and transduction. Viral vectors can bereplication-competent or replication-defective. In the case in whichviral replication is defective, replication will occur in host cellsproviding functions that complement the defects.

[0199] Vectors generally include selectable markers that enable theselection of the subpopulation of cells that contain the recombinantvector constructs. The marker can be contained in the same vector thatcontains the nucleic acid molecules described herein or may be on aseparate vector. Markers include tetracycline or ampicillin-resistancegenes for prokaryotic host cells and dihydrofolate reductase or neomycinresistance for eukaryotic host cells. However, any marker that providesselection for a phenotypic trait will be effective.

[0200] While the mature proteins can be produced in bacteria, yeast,mammalian cells, and other cells under the control of the appropriateregulatory sequences, cell-free transcription and translation systemscan also be used to produce these proteins using RNA derived from theDNA constructs described herein.

[0201] Where secretion of the peptide is desired, appropriate secretionsignals are incorporated into the vector. The signal sequence can beendogenous to the peptides or heterologous to these peptides.

[0202] Where the peptide is not secreted into the medium, the proteincan be isolated from the host cell by standard disruption procedures,including freeze thaw, sonication, mechanical disruption, use of lysingagents and the like. The peptide can then be recovered and purified bywell-known purification methods including ammonium sulfateprecipitation, acid extraction, anion or cationic exchangechromatography, phosphocellulose chromatography, hydrophobic-interactionchromatography, affinity chromatography, hydroxylapatite chromatography,lectin chromatography, or high performance liquid chromatography.

[0203] It is also understood that depending upon the host cell inrecombinant production of the peptides described herein, the peptidescan have various glycosylation patterns, depending upon the cell, ormaybe non-glycosylated as when produced in bacteria. In addition, thepeptides may include an initial modified methionine in some cases as aresult of a host-mediated process.

[0204] Uses of Vectors and Host Cells

[0205] The recombinant host cells expressing the peptides describedherein have a variety of uses. First, the cells are useful for producinga drug-metabolizing enzyme protein or peptide that can be furtherpurified to produce desired amounts of drug-metabolizing enzyme proteinor fragments. Thus, host cells containing expression vectors are usefulfor peptide production.

[0206] Host cells are also useful for conducting cell-based assaysinvolving the drug-metabolizing enzyme protein or drug-metabolizingenzyme protein fragments, such as those described above as well as otherformats known in the art. Thus, a recombinant host cell expressing anative drug-metabolizing enzyme protein is useful for assaying compoundsthat stimulate or inhibit drug-metabolizing enzyme protein function.

[0207] Host cells are also useful for identifying drug-metabolizingenzyme protein mutants in which these functions are affected. If themutants naturally occur and give rise to a pathology, host cellscontaining the mutations are useful to assay compounds that have adesired effect on the mutant drug-metabolizing enzyme protein (forexample, stimulating or inhibiting function) which may not be indicatedby their effect on the native drug-metabolizing enzyme protein.

[0208] Genetically engineered host cells can be further used to producenon-human transgenic animals. A transgenic animal is preferably amammal, for example a rodent, such as a rat or mouse, in which one ormore of the cells of the animal include a transgene. A transgene isexogenous DNA which is integrated into the genome of a cell from which atransgenic animal develops and which remains in the genome of the matureanimal in one or more cell types or tissues of the transgenic animal.These animals are useful for studying the function of adrug-metabolizing enzyme protein and identifying and evaluatingmodulators of drug-metabolizing enzyme protein activity. Other examplesof transgenic animals include non-human primates, sheep, dogs, cows,goats, chickens, and amphibians.

[0209] A transgenic animal can be produced by introducing nucleic acidinto the male pronuclei of a fertilized oocyte, e.g., by microinjection,retroviral infection, and allowing the oocyte to develop in apseudopregnant female foster animal. Any of the drug-metabolizing enzymeprotein nucleotide sequences can be introduced as a transgene into thegenome of a non-human animal, such as a mouse.

[0210] Any of the regulatory or other sequences useful in expressionvectors can form part of the transgenic sequence. This includes intronicsequences and polyadenylation signals, if not already included. Atissue-specific regulatory sequence(s) can be operably linked to thetransgene to direct expression of the drug-metabolizing enzyme proteinto particular cells.

[0211] Methods for generating transgenic animals via embryo manipulationand microinjection, particularly animals such as mice, have becomeconventional in the art and are described, for example, in U.S. Pat.Nos. 4,736,866 and 4,870,009, both by Leder et al., U.S. Pat. No.4,873,191 by Wagner et al. and in Hogan, B., Manipulating the MouseEmbryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,1986). Similar methods are used for production of other transgenicanimals. A transgenic founder animal can be identified based upon thepresence of the transgene in its genome and/or expression of transgenicmRNA in tissues or cells of the animals. A transgenic founder animal canthen be used to breed additional animals carrying the transgene.Moreover, transgenic animals carrying a transgene can further be bred toother transgenic animals carrying other transgenes. A transgenic animalalso includes animals in which the entire animal or tissues in theanimal have been produced using the homologously recombinant host cellsdescribed herein.

[0212] In another embodiment, transgenic non-human animals can beproduced which contain selected systems that allow for regulatedexpression of the transgene. One example of such a system is thecre/loxP recombinase system of bacteriophage P1. For a description ofthe cre/loxP recombinase system, see, e.g., Lakso et al. PNAS89:6232-6236 (1992). Another example of a recombinase system is the FLPrecombinase system of S. cerevisiae (O'Gorman et al. Science251:1351-1355 (1991). If a cre/loxP recombinase system is used toregulate expression of the transgene, animals containing transgenesencoding both the Cre recombinase and a selected protein is required.Such animals can be provided through the construction of “double”transgenic animals, e.g., by mating two transgenic animals, onecontaining a transgene encoding a selected protein and the othercontaining a transgene encoding a recombinase.

[0213] Clones of the non-human transgenic animals described herein canalso be produced according to the methods described in Wilmut, I. et al.Nature 385:810-813 (1997) and PCT International Publication Nos. WO97/07668 and WO 97/07669. In brief, a cell, e.g., a somatic cell, fromthe transgenic animal can be isolated and induced to exit the growthcycle and enter G_(o) phase. The quiescent cell can then be fused, e.g.,through the use of electrical pulses, to an enucleated oocyte from ananimal of the same species from which the quiescent cell is isolated.The reconstructed oocyte is then cultured such that it develops tomorula or blastocyst and then transferred to pseudopregnant femalefoster animal. The offspring born of this female foster animal will be aclone of the animal from which the cell, e.g., the somatic cell, isisolated.

[0214] Transgenic animals containing recombinant cells that express thepeptides described herein are useful to conduct the assays describedherein in an in vivo context. Accordingly, the various physiologicalfactors that are present in vivo and that could effect substratebinding, drug-metabolizing enzyme protein activation, and signaltransduction, may not be evident from in vitro cell-free or cell-basedassays. Accordingly, it is useful to provide non-human transgenicanimals to assay in vivo drug-metabolizing enzyme protein function,including substrate interaction, the effect of specific mutantdrug-metabolizing enzyme proteins on drug-metabolizing enzyme proteinfunction and substrate interaction, and the effect of chimericdrug-metabolizing enzyme proteins. It is also possible to assess theeffect of null mutations, that is mutations that substantially orcompletely eliminate one or more drug-metabolizing enzyme proteinfunctions.

[0215] All publications and patents mentioned in the above specificationare herein incorporated by reference. Various modifications andvariations of the described method and system of the invention will beapparent to those skilled in the art without departing from the scopeand spirit of the invention. Although the invention has been describedin connection with specific preferred embodiments, it should beunderstood that the invention as claimed should not be unduly limited tosuch specific embodiments. Indeed, various modifications of theabove-described modes for carrying out the invention which are obviousto those skilled in the field of molecular biology or related fields areintended to be within the scope of the following claims.

1 4 1 1413 DNA Homo sapiens 1 agaatgatca cattgcacca ggatgactctgaaatggact tcagttctcc tgctgataca 60 tctcagttgt tactttagct ctgggagttgtggaaaagtg ctggtgtggg ccgcagaata 120 cagccattgg atgaatatga agacaatcctgaaagagctt gttcagagag gtcatgaggt 180 gactgtactg gcatcttcag cttccattctttttgatccc aatgatgcat ccactcttaa 240 atttgaagtt tatcctacat ctttaactaaaactgaattt gagaatatca tcatgcaaca 300 ggttaagaga tggtcagaca ttcgaaaagatagcttttgg ttatattttt cacaagaaca 360 agaaatcctg tgggaattat atgacatatttagaaacttc tgtaaagatg tagtttcaaa 420 taagaaagtt atgaaaaaac tacaagagttaagatttgac atcgtttttg cagatgctgt 480 ttttccctgt ggtgagctgc tggctgcgctacttaacata cgacccacta ccttatttga 540 gacaatggga aaagctgaca tatggcttatgcgaaacccc tggagttttc aatttcctca 600 tccattctta ccaaacgttg attttgttggaggattccac tgcaaacctg ccaaacccct 660 acctaaggaa atggaggagt ttgtacagagctctggagaa aatggtgttg tggtgttttc 720 tctggggtca gtgataagta acatgacagcagaaagggcc aatgtaattg caacagccct 780 tgccaggatc ccacaaaagg ttctgtggagatttgacggg aataaaccag atgccttagg 840 tctcaatact cggctgtaca agtggataccccagaatgac cttctaggtc atccaaaaac 900 cagagctttt ataactcatg gtggagccaatggcatctat gaggcaatct accatgggat 960 ccccatggtg ggcattccat tgttttttgatcaacctgat aacattgctc acatgaaggc 1020 caagggagca gctgttagat tggacttcaacacaatgtcg agtacagacc tgctgaatgc 1080 actgaagaca gtaattaatg atcctttatataaagagaat attatgaaat tatcaagaat 1140 tcaacatgat caaccagtaa agcccctggatcgagcagtc ttctggattg aatttgtcat 1200 gccccacaaa ggagccaaac accttcgagttgcagcccat gacctcacct ggttccagta 1260 ccactctttg gatgtgattg ggtttctgctggcctgtgtg gcaactgtga tatttatcat 1320 cacaaagttt tgtctgtttt gtttctggaagtttgctaga aaagggaaga agggaaaaag 1380 agattagtta tgtccgacat ttgaagctgaagg 1413 2 454 PRT Homo sapiens 2 Met Thr Leu Lys Trp Thr Ser Val LeuLeu Leu Ile His Leu Ser Cys 1 5 10 15 Tyr Phe Ser Ser Gly Ser Cys GlyLys Val Leu Val Trp Ala Ala Glu 20 25 30 Tyr Ser His Trp Met Asn Met LysThr Ile Leu Lys Glu Leu Val Gln 35 40 45 Arg Gly His Glu Val Thr Val LeuAla Ser Ser Ala Ser Ile Leu Phe 50 55 60 Asp Pro Asn Asp Ala Ser Thr LeuLys Phe Glu Val Tyr Pro Thr Ser 65 70 75 80 Leu Thr Lys Thr Glu Phe GluAsn Ile Ile Met Gln Gln Val Lys Arg 85 90 95 Trp Ser Asp Ile Arg Lys AspSer Phe Trp Leu Tyr Phe Ser Gln Glu 100 105 110 Gln Glu Ile Leu Trp GluLeu Tyr Asp Ile Phe Arg Asn Phe Cys Lys 115 120 125 Asp Val Val Ser AsnLys Lys Val Met Lys Lys Leu Gln Glu Leu Arg 130 135 140 Phe Asp Ile ValPhe Ala Asp Ala Val Phe Pro Cys Gly Glu Leu Leu 145 150 155 160 Ala AlaLeu Leu Asn Ile Arg Pro Thr Thr Leu Phe Glu Thr Met Gly 165 170 175 LysAla Asp Ile Trp Leu Met Arg Asn Pro Trp Ser Phe Gln Phe Pro 180 185 190His Pro Phe Leu Pro Asn Val Asp Phe Val Gly Gly Phe His Cys Lys 195 200205 Pro Ala Lys Pro Leu Pro Lys Glu Met Glu Glu Phe Val Gln Ser Ser 210215 220 Gly Glu Asn Gly Val Val Val Phe Ser Leu Gly Ser Val Ile Ser Asn225 230 235 240 Met Thr Ala Glu Arg Ala Asn Val Ile Ala Thr Ala Leu AlaArg Ile 245 250 255 Pro Gln Lys Val Leu Trp Arg Phe Asp Gly Asn Lys ProAsp Ala Leu 260 265 270 Gly Leu Asn Thr Arg Leu Tyr Lys Trp Ile Pro GlnAsn Asp Leu Leu 275 280 285 Gly His Pro Lys Thr Arg Ala Phe Ile Thr HisGly Gly Ala Asn Gly 290 295 300 Ile Tyr Glu Ala Ile Tyr His Gly Ile ProMet Val Gly Ile Pro Leu 305 310 315 320 Phe Phe Asp Gln Pro Asp Asn IleAla His Met Lys Ala Lys Gly Ala 325 330 335 Ala Val Arg Leu Asp Phe AsnThr Met Ser Ser Thr Asp Leu Leu Asn 340 345 350 Ala Leu Lys Thr Val IleAsn Asp Pro Leu Tyr Lys Glu Asn Ile Met 355 360 365 Lys Leu Ser Arg IleGln His Asp Gln Pro Val Lys Pro Leu Asp Arg 370 375 380 Ala Val Phe TrpIle Glu Phe Val Met Pro His Lys Gly Ala Lys His 385 390 395 400 Leu ArgVal Ala Ala His Asp Leu Thr Trp Phe Gln Tyr His Ser Leu 405 410 415 AspVal Ile Gly Phe Leu Leu Ala Cys Val Ala Thr Val Ile Phe Ile 420 425 430Ile Thr Lys Phe Cys Leu Phe Cys Phe Trp Lys Phe Ala Arg Lys Gly 435 440445 Lys Lys Gly Lys Arg Asp 450 3 288 PRT Homo sapiens 3 Arg Pro Thr ThrLeu Phe Glu Thr Met Gly Lys Ala Asp Ile Trp Leu 1 5 10 15 Met Arg AsnSer Trp Ser Phe Gln Phe Pro His Pro Phe Leu Pro Asn 20 25 30 Val Asp PheVal Gly Gly Phe His Cys Lys Pro Ala Lys Pro Leu Pro 35 40 45 Lys Glu MetGlu Glu Phe Val Gln Ser Ser Gly Glu Asn Gly Val Val 50 55 60 Val Phe SerLeu Gly Ser Val Ile Ser Asn Met Thr Ala Glu Arg Ala 65 70 75 80 Asn ValIle Ala Thr Ala Leu Ala Lys Ile Pro Gln Lys Val Leu Trp 85 90 95 Arg PheAsp Gly Asn Lys Pro Asp Ala Leu Gly Leu Asn Thr Arg Leu 100 105 110 TyrLys Trp Ile Pro Gln Asn Asp Leu Leu Gly His Pro Lys Thr Arg 115 120 125Ala Phe Ile Thr His Gly Gly Ala Asn Gly Ile Tyr Glu Ala Ile Tyr 130 135140 His Gly Ile Pro Met Val Gly Ile Pro Leu Phe Phe Asp Gln Pro Asp 145150 155 160 Asn Ile Ala His Met Lys Ala Lys Gly Ala Ala Val Arg Leu AspPhe 165 170 175 Asn Thr Met Ser Ser Thr Asp Leu Leu Asn Ala Leu Lys ThrVal Ile 180 185 190 Asn Asp Pro Leu Tyr Lys Glu Asn Ile Met Lys Leu SerArg Ile Gln 195 200 205 His Asp Gln Pro Val Lys Pro Leu Asp Arg Ala ValPhe Trp Ile Glu 210 215 220 Phe Val Met Pro His Lys Gly Ala Lys His LeuArg Val Ala Ala His 225 230 235 240 Asp Leu Thr Trp Phe Gln Tyr His SerLeu Asp Val Ile Gly Phe Leu 245 250 255 Leu Ala Cys Val Ala Thr Val IlePhe Ile Ile Thr Lys Phe Cys Leu 260 265 270 Phe Cys Phe Trp Lys Phe AlaArg Lys Gly Lys Lys Gly Lys Arg Asp 275 280 285 4 197 PRT Homo sapiens 4Met Thr Leu Lys Trp Thr Ser Val Leu Leu Leu Ile His Leu Ser Cys 1 5 1015 Tyr Phe Ser Ser Gly Ser Cys Gly Lys Val Leu Val Trp Ala Ala Glu 20 2530 Tyr Ser His Trp Met Asn Met Lys Thr Ile Leu Lys Glu Leu Val Gln 35 4045 Arg Gly His Glu Val Thr Val Leu Ala Ser Ser Ala Ser Ile Leu Phe 50 5560 Asp Pro Asn Asp Ala Ser Thr Leu Lys Phe Glu Val Tyr Pro Thr Ser 65 7075 80 Leu Thr Lys Thr Glu Phe Glu Asn Ile Ile Met Gln Gln Val Lys Arg 8590 95 Trp Ser Asp Ile Arg Lys Asp Ser Phe Trp Leu Tyr Phe Ser Gln Glu100 105 110 Gln Glu Ile Leu Trp Glu Leu Tyr Asp Ile Phe Arg Asn Phe CysLys 115 120 125 Asp Val Val Ser Asn Lys Lys Val Met Lys Lys Leu Gln GluSer Arg 130 135 140 Phe Asp Ile Val Phe Ala Asp Ala Val Phe Pro Cys GlyGlu Leu Leu 145 150 155 160 Ala Ala Leu Leu Asn Ile Arg Phe Val Tyr SerLeu Arg Phe Thr Pro 165 170 175 Gly Tyr Thr Ile Glu Arg His Ser Gly GlyLeu Ile Phe Pro Pro Ser 180 185 190 Tyr Ile Pro Ile Val 195

That which is claimed is:
 1. An isolated nucleic acid molecule encodinga UDP-glycosyltransferase, wherein the isolated nucleic acid moleculecomprises a nucleotide sequence selected from the group consisting of:(a) a nucleotide sequence that encodes a protein comprising the aminoacid sequence of SEQ ID NO:2; and (b) a nucleotide sequence consistingof SEQ ID NO:1.
 2. A nucleic acid vector comprising a nucleic acidmolecule of claim
 1. 3. A host cell containing the vector of claim
 2. 4.A process for producing a polypeptide comprising culturing the host cellof claim 3 under conditions sufficient for the production of saidpolypeptide, and recovering the peptide from the host cell culture. 5.An isolated polynucleotide comprising a nucleotide sequence set forth inSEQ ID NO:1.
 6. A vector according to claim 2, wherein said vector isselected from the group consisting of a plasmid, virus, andbacteriophage.
 7. A vector according to claim 6, wherein said isolatednucleic acid molecule is inserted into said vector in proper orientationand correct reading frame such that the protein of SEQ ID NO:2 may beexpressed by a cell transformed with said vector.
 8. A vector accordingto claim 7, wherein said isolated nucleic acid molecule is operativelylinked to a promoter sequence.
 9. An isolated nucleic acid moleculecomprising a nucleotide sequence that is completely complementary to anucleotide sequence of claim
 1. 10. An isolated nucleic acid moleculeencoding a UDP-glycosyltransferase, wherein the isolated nucleic acidmolecule comprises a nucleotide sequence selected from the groupconsisting of: (a) a nucleotide sequence that encodes a proteincomprising an amino acid sequence having at least 95% sequence identityto SEQ ID NO:2; and (b) a nucleotide sequence having at least 95%sequence identity to SEQ ID NO:1.
 11. An isolated nucleic acid moleculecomprising a nucleotide sequence that is completely complementary to anucleotide sequence of claim 10.